Publications


Quantification of flagellar motor stator dynamics through in vivo proton-motive force control.

Mol Microbiol 87 (2013) 338-347

MJ Tipping, BC Steel, NJ Delalez, RM Berry, JP Armitage

The bacterial flagellar motor, one of the few rotary motors in nature, produces torque to drive the flagellar filament by ion translocation through membrane-bound stator complexes. We used the light-driven proton pump proteorhodopsin (pR) to control the proton-motive force (PMF) in vivo by illumination. pR excitation was shown to be sufficient to replace native PMF generation, and when excited in cells with intact native PMF generation systems increased motor speed beyond the physiological norm. We characterized the effects of rapid in vivo PMF changes on the flagellar motor. Transient PMF disruption events from loss of illumination caused motors to stop, with rapid recovery of their previous rotation rate after return of illumination. However, extended periods of PMF loss led to stepwise increases in rotation rate upon PMF return as stators returned to the motor. The rate constant for stator binding to a putative single binding site on the motor was calculated to be 0.06 s(-1). Using GFP-tagged MotB stator proteins, we found that transient PMF disruption leads to reversible stator diffusion away from the flagellar motor, showing that PMF presence is necessary for continued motor integrity, and calculated a stator dissociation rate of 0.038 s(-1).


Investigating Stator Dynamics of the Escherichia Coli Flagellar Motor

BIOPHYSICAL JOURNAL 104 (2013) 640A-640A

LE Dickinson, MM van Oene, F Pedaci, B Cross, R Lim, RM Berry, NH Dekker


High-resolution single-molecule characterization of the enzymatic states in Escherichia coli F1-ATPase.

Philosophical transactions of the Royal Society of London. Series B, Biological sciences 368 (2013) 20120023-

T Bilyard, M Nakanishi-Matsui, BC Steel, T Pilizota, AL Nord, H Hosokawa, M Futai, RM Berry

The rotary motor F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)) is one of the best-studied of all molecular machines. F(1)-ATPase is the part of the enzyme F(1)F(O)-ATP synthase that is responsible for generating most of the ATP in living cells. Single-molecule experiments have provided a detailed understanding of how ATP hydrolysis and synthesis are coupled to internal rotation within the motor. In this work, we present evidence that mesophilic F(1)-ATPase from Escherichia coli (EF(1)) is governed by the same mechanism as TF(1) under laboratory conditions. Using optical microscopy to measure rotation of a variety of marker particles attached to the γ-subunit of single surface-bound EF(1) molecules, we characterized the ATP-binding, catalytic and inhibited states of EF(1). We also show that the ATP-binding and catalytic states are separated by 35±3°. At room temperature, chemical processes occur faster in EF(1) than in TF(1), and we present a methodology to compensate for artefacts that occur when the enzymatic rates are comparable to the experimental temporal resolution. Furthermore, we show that the molecule-to-molecule variation observed at high ATP concentration in our single-molecule assays can be accounted for by variation in the orientation of the rotating markers.


Flagellar hook flexibility is essential for bundle formation in swimming Escherichia coli cells.

J Bacteriol 194 (2012) 3495-3501

MT Brown, BC Steel, C Silvestrin, DA Wilkinson, NJ Delalez, CN Lumb, B Obara, JP Armitage, RM Berry

Swimming Escherichia coli cells are propelled by the rotary motion of their flagellar filaments. In the normal swimming pattern, filaments positioned randomly over the cell form a bundle at the posterior pole. It has long been assumed that the hook functions as a universal joint, transmitting rotation on the motor axis through up to ∼90° to the filament in the bundle. Structural models of the hook have revealed how its flexibility is expected to arise from dynamic changes in the distance between monomers in the helical lattice. In particular, each of the 11 protofilaments that comprise the hook is predicted to cycle between short and long forms, corresponding to the inside and outside of the curved hook, once each revolution of the motor when the hook is acting as a universal joint. To test this, we genetically modified the hook so that it could be stiffened by binding streptavidin to biotinylated monomers, impeding their motion relative to each other. We found that impeding the action of the universal joint resulted in atypical swimming behavior as a consequence of disrupted bundle formation, in agreement with the universal joint model.


Conformational spread in the flagellar motor switch: A model study

PLoS Computational Biology 8 (2012)

Q Ma, DV Nicolau, PK Maini, RM Berry, F Bai

The reliable response to weak biological signals requires that they be amplified with fidelity. In E. coli, the flagellar motors that control swimming can switch direction in response to very small changes in the concentration of the signaling protein CheY-P, but how this works is not well understood. A recently proposed allosteric model based on cooperative conformational spread in a ring of identical protomers seems promising as it is able to qualitatively reproduce switching, locked state behavior and Hill coefficient values measured for the rotary motor. In this paper we undertook a comprehensive simulation study to analyze the behavior of this model in detail and made predictions on three experimentally observable quantities: switch time distribution, locked state interval distribution, Hill coefficient of the switch response. We parameterized the model using experimental measurements, finding excellent agreement with published data on motor behavior. Analysis of the simulated switching dynamics revealed a mechanism for chemotactic ultrasensitivity, in which cooperativity is indispensable for realizing both coherent switching and effective amplification. These results showed how cells can combine elements of analog and digital control to produce switches that are simultaneously sensitive and reliable. © 2012 Ma et al.


The microbial olympics

Nature Reviews Microbiology 10 (2012) 583-588

M Youle, F Rohwer, A Stacy, M Whiteley, BC Steel, NJ Delalez, AL Nord, RM Berry, JP Armitage, S Kamoun, S Hogenhout, SP Diggle, J Gurney, EJG Pollitt, A Boetius, SC Cary

Every four years, the Olympic Games plays host to competitors who have built on their natural talent by training for many years to become the best in their chosen discipline. Similar spirit and endeavour can be found throughout the microbial world, in which every day is a competition to survive and thrive. Microorganisms are trained through evolution to become the fittest and the best adapted to a particular environmental niche or lifestyle, and to innovate when the 'rules of the game' are changed by alterations to their natural habitats. In this Essay, we honour the best competitors in the microbial world by inviting them to take part in the inaugural Microbial Olympics. © 2012 Macmillan Publishers Limited. All rights reserved.


The rotary bacterial flagellar motor

in Comprehensive Biophysics, 8 (2012) 50-71

Y Sowa, RM Berry

Bacterial cell envelopes often contain a flagellar motor - a reversible rotary nanomachine with an approximate diameter of 45nm - that allows cells to swim. Power is provided by the movement of H+ or Na+ down the electrochemical gradients across the cytoplasmic membrane, often termed the proton motive force or sodium motive force. A helical filament is rotated by each motor at several hundred revolutions per second. In many species, the motor switches direction stochastically; switching rates are controlled by a network of sensory and signaling proteins. The first direct observation, approximately 40 years ago, of the function of a single molecular motor was of the bacterial flagellar motor. Nevertheless, due to the large size and complexity of the motor, much remains to be discovered about this nanomachine, particularly the many structural details of the torque-generating mechanism. This chapter summarizes what has been learned about the structure and function of the motor with a focus on recent observations, particularly those obtained using single molecule techniques. © 2012 Elsevier B.V. All rights reserved.


Erratum: The microbial olympics (Nature Reviews Microbiology (2012) 10 (583-588))

Nature Reviews Microbiology 10 (2012) 654-

M Youle, F Rohwer, A Stacy, M Whiteley, BC Steel, NJ Delalez, AL Nord, RM Berry, JP Armitage, S Kamoun, S Hogenhout, SP Diggle, J Gurney, EJG Pollitt, A Boetius, C Cary


Steps and bumps: Precision extraction of discrete states of molecular machines

Biophysical Journal 101 (2011) 477-485

MA Little, BC Steel, F Bai, Y Sowa, T Bilyard, DM Mueller, RM Berry, NS Jones

We report statistical time-series analysis tools providing improvements in the rapid, precision extraction of discrete state dynamics from time traces of experimental observations of molecular machines. By building physical knowledge and statistical innovations into analysis tools, we provide techniques for estimating discrete state transitions buried in highly correlated molecular noise. We demonstrate the effectiveness of our approach on simulated and real examples of steplike rotation of the bacterial flagellar motor and the F1-ATPase enzyme. We show that our method can clearly identify molecular steps, periodicities and cascaded processes that are too weak for existing algorithms to detect, and can do so much faster than existing algorithms. Our techniques represent a step in the direction toward automated analysis of high-sample-rate, molecular-machine dynamics. Modular, open-source software that implements these techniques is provided. © 2011 Biophysical Society.


Two methods of temperature control for single-molecule measurements.

Eur Biophys J 40 (2011) 651-660

MAB Baker, Y Inoue, K Takeda, A Ishijima, RM Berry

Modern single-molecule biophysical experiments require high numerical aperture oil-immersion objectives in close contact with the sample. We introduce two methods of high numerical aperture temperature control which can be implemented on any microscope: objective temperature control using a ring-shaped Peltier device, and stage temperature control using a fluid flow cooling chip in close thermal contact with the sample. We demonstrate the efficacy of each system by showing the change in speed with temperature of two molecular motors, the bacterial flagellar motor and skeletal muscle myosin.


Myxobacteria gliding motility requires cytoskeleton rotation powered by proton motive force

Proceedings of the National Academy of Sciences of the United States of America 108 (2011) 2498-2503

B Nan, J Chen, JC Neu, RM Berry, G Oster, DR Zusman

Myxococcus xanthus is a Gram-negative bacterium that glides over surfaces without the aid of flagella. Two motility systems are used for locomotion: social-motility, powered by the retraction of type IV pili, and adventurous (A)-motility, powered by unknown mechanism(s). We have shown that AgmU, an A-motility protein, is part of a multiprotein complex that spans the inner membrane and periplasm of M. xanthus. In this paper, we present evidence that periplasmic AgmU decorates a looped continuous helix that rotates clockwise as cells glide forward, reversing its rotation when cells reverse polarity. Inhibitor studies showed that the AgmU helix rotation is driven by proton motive force (PMF) and depends on actin-like MreB cytoskeletal filaments. The AgmU motility complex was found to interact with MotAB homologs. Our data are consistent with a mechanochemical model in which PMF-driven motors, similar to bacterial flagella stator complexes, run along an endless looped helical track, driving rotation of the track; deformation of the cell surface by the AgmU-associated proteins creates pressure waves in the slime, pushing cells forward.


Steps and bumps: precision extraction of discrete states of molecular machines.

Biophys J 101 (2011) 477-485

MA Little, BC Steel, F Bai, Y Sowa, T Bilyard, DM Mueller, RM Berry, NS Jones

We report statistical time-series analysis tools providing improvements in the rapid, precision extraction of discrete state dynamics from time traces of experimental observations of molecular machines. By building physical knowledge and statistical innovations into analysis tools, we provide techniques for estimating discrete state transitions buried in highly correlated molecular noise. We demonstrate the effectiveness of our approach on simulated and real examples of steplike rotation of the bacterial flagellar motor and the F1-ATPase enzyme. We show that our method can clearly identify molecular steps, periodicities and cascaded processes that are too weak for existing algorithms to detect, and can do so much faster than existing algorithms. Our techniques represent a step in the direction toward automated analysis of high-sample-rate, molecular-machine dynamics. Modular, open-source software that implements these techniques is provided.


Proteins of Functioning Flagellar Rotor Turnover but only in the Presence of Signalling Proteins

BIOPHYSICAL JOURNAL 98 (2010) 433A-433A

NJ Delalez, GH Wadhams, RM Berry, JP Armitage, MC Leake


Conformational spread as a mechanism for cooperativity in the bacterial flagellar switch.

Science 327 (2010) 685-689

F Bai, RW Branch, DV Nicolau, T Pilizota, BC Steel, PK Maini, RM Berry

The bacterial flagellar switch that controls the direction of flagellar rotation during chemotaxis has a highly cooperative response. This has previously been understood in terms of the classic two-state, concerted model of allosteric regulation. Here, we used high-resolution optical microscopy to observe switching of single motors and uncover the stochastic multistate nature of the switch. Our observations are in detailed quantitative agreement with a recent general model of allosteric cooperativity that exhibits conformational spread--the stochastic growth and shrinkage of domains of adjacent subunits sharing a particular conformational state. We expect that conformational spread will be important in explaining cooperativity in other large signaling complexes.


Single Molecule Rotation of F1-ATPase from S. cerevisiae

BIOPHYSICAL JOURNAL 98 (2010) 433A-434A

BC Steel, Y Wang, V Pagadala, RM Berry, DM Mueller


Time for bacteria to slow down.

Cell 141 (2010) 24-26

JP Armitage, RM Berry

The speed of the bacterial flagellar motor is thought to be regulated by structural changes in the motor. Two new studies, Boehm et al. (2010) in this issue and Paul et al. (2010) in Molecular Cell, now show that cyclic di-GMP also regulates flagellar motor speed through interactions between the cyclic di-GMP binding protein YcgR and the motor proteins.


Signal-dependent turnover of the bacterial flagellar switch protein FliM

Proceedings of the National Academy of Sciences of the United States of America 107 (2010) 11347-11351

NJ Delalez, GH Wadhams, G Rosser, Q Xue, MT Brown, IM Dobbie, RM Berry, MC Leake, JP Armitage

Most biological processes are performed by multiprotein complexes. Traditionally described as static entities, evidence is now emerging that their components can be highly dynamic, exchanging constantly with cellular pools. The bacterial flagellar motor contains ∼13 different proteins and provides an ideal system to study functional molecular complexes. It is powered by transmembrane ion flux through a ring of stator complexes that push on a central rotor. The Escherichia coli motor switches direction stochastically in response to binding of the response regulator CheY to the rotor switch component FliM. Much is known of the static motor structure, but we are just beginning to understand the dynamics of its individual components. Here we measure the stoichiometry and turnover of FliM in functioning flagellar motors, by using high-resolution fluorescence microscopy of E. coli expressing genomically encoded YPet derivatives of FliM at physiological levels. We show that the ∼30 FliM molecules per motor exist in two discrete populations, one tightly associated with the motor and the other undergoing stochastic turnover. This turnover of FliM molecules depends on the presence of active CheY, suggesting a potential role in the process of motor switching. In many ways the bacterial flagellar motor is as an archetype macromolecular assembly, and our results may have further implications for the functional relevance of protein turnover in other large molecular complexes.


Time for Bacteria to Slow down

Cell 141 (2010) 24-26

JP Armitage, RM Berry

The speed of the bacterial flagellar motor is thought to be regulated by structural changes in the motor. Two new studies, Boehm et al. (2010) in this issue and Paul et al. (2010) in Molecular Cell, now show that cyclic di-GMP also regulates flagellar motor speed through interactions between the cyclic di-GMP binding protein YcgR and the motor proteins. © 2010 Elsevier Inc.


A simple backscattering microscope for fast tracking of biological molecules.

Rev Sci Instrum 81 (2010) 113704-

Y Sowa, BC Steel, RM Berry

Recent developments in techniques for observing single molecules under light microscopes have helped reveal the mechanisms by which molecular machines work. A wide range of markers can be used to detect molecules, from single fluorophores to micron sized markers, depending on the research interest. Here, we present a new and simple objective-type backscattering microscope to track gold nanoparticles with nanometer and microsecond resolution. The total noise of our system in a 55 kHz bandwidth is ~0.6 nm per axis, sufficient to measure molecular movement. We found our backscattering microscopy to be useful not only for in vitro but also for in vivo experiments because of lower background scattering from cells than in conventional dark-field microscopy. We demonstrate the application of this technique to measuring the motion of a biological rotary molecular motor, the bacterial flagellar motor, in live Escherichia coli cells.


Signal-dependent turnover of the bacterial flagellar switch protein FliM.

Proc Natl Acad Sci U S A 107 (2010) 11347-11351

NJ Delalez, GH Wadhams, G Rosser, Q Xue, MT Brown, IM Dobbie, RM Berry, MC Leake, JP Armitage

Most biological processes are performed by multiprotein complexes. Traditionally described as static entities, evidence is now emerging that their components can be highly dynamic, exchanging constantly with cellular pools. The bacterial flagellar motor contains approximately 13 different proteins and provides an ideal system to study functional molecular complexes. It is powered by transmembrane ion flux through a ring of stator complexes that push on a central rotor. The Escherichia coli motor switches direction stochastically in response to binding of the response regulator CheY to the rotor switch component FliM. Much is known of the static motor structure, but we are just beginning to understand the dynamics of its individual components. Here we measure the stoichiometry and turnover of FliM in functioning flagellar motors, by using high-resolution fluorescence microscopy of E. coli expressing genomically encoded YPet derivatives of FliM at physiological levels. We show that the approximately 30 FliM molecules per motor exist in two discrete populations, one tightly associated with the motor and the other undergoing stochastic turnover. This turnover of FliM molecules depends on the presence of active CheY, suggesting a potential role in the process of motor switching. In many ways the bacterial flagellar motor is as an archetype macromolecular assembly, and our results may have further implications for the functional relevance of protein turnover in other large molecular complexes.

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