Publications by Richard Berry


Load-dependent adaptation near zero load in the bacterial flagellar motor.

Journal of the Royal Society, Interface 16 (2019) ARTN 20190300

RM Berry, AL Nord, JA Nirody

The bacterial flagellar motor is an ion-powered transmembrane protein complex which drives swimming in many bacterial species. The motor consists of a cytoplasmic 'rotor' ring and a number of 'stator' units, which are bound to the cell wall of the bacterium. Recently, it has been shown that the number of functional torque-generating stator units in the motor depends on the external load, and suggested that mechanosensing in the flagellar motor is driven via a 'catch bond' mechanism in the motor's stator units. We present a method that allows us to measure-on a single motor-stator unit dynamics across a large range of external loads, including near the zero-torque limit. By attaching superparamagnetic beads to the flagellar hook, we can control the motor's speed via a rotating magnetic field. We manipulate the motor to four different speed levels in two different ion-motive force (IMF) conditions. This framework allows for a deeper exploration into the mechanism behind load-dependent remodelling by separating out motor properties, such as rotation speed and energy availability in the form of IMF, that affect the motor torque.


Simultaneous Tracking of Pseudomonas aeruginosa Motility in Liquid and at the Solid-Liquid Interface Reveals Differential Roles for the Flagellar Stators.

mSystems 4 (2019)

AL Hook, JL Flewellen, J-F Dubern, AM Carabelli, IM Zaid, RM Berry, RD Wildman, N Russell, P Williams, MR Alexander

Bacteria sense chemicals, surfaces, and other cells and move toward some and away from others. Studying how single bacterial cells in a population move requires sophisticated tracking and imaging techniques. We have established quantitative methodology for label-free imaging and tracking of individual bacterial cells simultaneously within the bulk liquid and at solid-liquid interfaces by utilizing the imaging modes of digital holographic microscopy (DHM) in three dimensions (3D), differential interference contrast (DIC), and total internal reflectance microscopy (TIRM) in two dimensions (2D) combined with analysis protocols employing bespoke software. To exemplify and validate this methodology, we investigated the swimming behavior of a Pseudomonas aeruginosa wild-type strain and isogenic flagellar stator mutants (motAB and motCD) within the bulk liquid and at the surface at the single-cell and population levels. Multiple motile behaviors were observed that could be differentiated by speed and directionality. Both stator mutants swam slower and were unable to adjust to the near-surface environment as effectively as the wild type, highlighting differential roles for the stators in adapting to near-surface environments. A significant reduction in run speed was observed for the P. aeruginosa mot mutants, which decreased further on entering the near-surface environment. These results are consistent with the mot stators playing key roles in responding to the near-surface environment.IMPORTANCE We have established a methodology to enable the movement of individual bacterial cells to be followed within a 3D space without requiring any labeling. Such an approach is important to observe and understand how bacteria interact with surfaces and form biofilm. We investigated the swimming behavior of Pseudomonas aeruginosa, which has two flagellar stators that drive its swimming motion. Mutants that had only either one of the two stators swam slower and were unable to adjust to the near-surface environment as effectively as the wild type. These results are consistent with the mot stators playing key roles in responding to the near-surface environment and could be used by bacteria to sense via their flagella when they are near a surface.


A multi-mode digital holographic microscope

Review of Scientific Instruments AIP Publishing 90 (2019) 023705-

J Flewellen, R Berry, I Zaid

We present a transmission-mode digital holographic microscope that can switch easily between three different imaging modes: inline, dark field off-axis, and bright field off-axis. Our instrument can be used: to track through time in three dimensions microscopic dielectric objects, such as motile micro-organisms; localize brightly scattering nanoparticles, which cannot be seen under conventional bright field illumination; and recover topographic information and measure the refractive index and dry mass of samples via quantitative phase recovery. Holograms are captured on a digital camera capable of high-speed video recording of up to 2000 frames per second. The inline mode of operation can be easily configurable to a large range of magnifications. We demonstrate the efficacy of the inline mode in tracking motile bacteria in three dimensions in a 160 μm × 160 μm × 100 μm volume at 45× magnification. Through the use of a novel physical mask in a conjugate Fourier plane in the imaging path, we use our microscope for high magnification, dark field off-axis holography, demonstrated by localizing 100 nm gold nanoparticles at 225× magnification up to at least 16 μm from the imaging plane. Finally, the bright field off-axis mode facilitates quantitative phase microscopy, which we employ to measure the refractive index of a standard resolution test target and to measure the dry mass of human erythrocytes.


Subunit exchange in protein complexes

Journal of Molecular Biology Elsevier 430 (2018) 4557-4579

S Tusk, N Delalez, R Berry

Over the past 50 years, protein complexes have been studied with techniques such as X-ray crystallography and electron microscopy, generating images which although detailed are static and homogeneous. More recently, limited application of in vivo fluorescence and other techniques has revealed that many complexes previously thought stable and compositionally uniform are dynamically variable, continually exchanging components with a freely circulating pool of “spares.” Here, we consider the purpose and prevalence of protein exchange, first reviewing the ongoing story of exchange in the bacterial flagella motor, before surveying reports of exchange in complexes across all domains of life, together highlighting great diversity in timescales and functions. Finally, we put this in the context of high-throughput proteomic studies which hint that exchange might be the norm, rather than an exception.


Imaging of Single Dye-Labeled Chemotaxis Proteins in Live Bacteria Using Electroporation.

Methods in molecular biology (Clifton, N.J.) 1729 (2018) 233-246

D Di Paolo, RM Berry

For the last 2 decades, the use of genetically fused fluorescent proteins (FPs) has greatly contributed to the study of chemotactic signaling in E. coli, including the activation of the response regulator protein CheY and its interaction with the flagellar motor. However, this approach suffers from a number of limitations, both biological and biophysical. For example, not all fusions are fully functional when fused to a bulky FP, which can have a similar molecular weight to its fused counterpart. FPs may interfere with the native interactions of the protein, and their chromophores have low brightness and photostability, and fast photobleaching rates. Electroporation allows for internalization of purified CheY proteins labeled with organic dyes into E. coli cells in controllable concentrations. Using fluorescence video microscopy, it is possible to observe single CheY molecules diffusing within cells and interacting with the sensory clusters and the flagellar motors in real time.


Detergent-free ultrafast reconstitution of membrane proteins into lipid bilayers using fusogenic complementary-charged proteoliposomes

Journal of Visualized Experiments Journal of Visualized Experiments 134 (2018) e56909

SB Vik, MA Galkin, A Russell, R Berry, R Ishmukhametov

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers are less compatible with detergents. Here we describe a strategy for bypassing this fundamental limitation using fusogenic oppositely charged liposomes bearing a membrane protein of interest. Fusion between such vesicles occurs within 5 min in a low ionic strength buffer. Positively charged fusogenic liposomes can be used as simple shuttle vectors for detergent-free delivery of membrane proteins into biomimetic target lipid bilayers, which are negatively charged. We also show how to reconstitute membrane proteins into fusogenic proteoliposomes with a fast 30-min protocol. Combining these two approaches, we demonstrate a fast assembly of an electron transport chain consisting of two membrane proteins from E. coli, a primary proton pump bo 3 -oxidase and F 1 F o ATP synthase, in membranes of vesicles of various sizes, ranging from 0.1 to > 10 microns, as well as ATP production by this chain.


A Catch-Bond Drives Stator Mechanosensitivity in the Bacterial Flagellar Motor

BIOPHYSICAL JOURNAL 114 (2018) 372A-372A

RM Berry, J Nirody, R Perez-Carrasco, A Barducci, F Pedaci, AL Nord, E Gachon


Torque Generation in the Bacterial Flagellar Motor

BIOPHYSICAL JOURNAL Elsevier BV 112 (2017) 30A-30A

JA Nirody, RM Berry, G Oster


Catch bond drives stator mechanosensitivity in the bacterial flagellar motor.

Proceedings of the National Academy of Sciences of the United States of America 114 (2017) 12952-12957

AL Nord, E Gachon, R Perez-Carrasco, JA Nirody, A Barducci, RM Berry, F Pedaci

The bacterial flagellar motor (BFM) is the rotary motor that rotates each bacterial flagellum, powering the swimming and swarming of many motile bacteria. The torque is provided by stator units, ion motive force-powered ion channels known to assemble and disassemble dynamically in the BFM. This turnover is mechanosensitive, with the number of engaged units dependent on the viscous load experienced by the motor through the flagellum. However, the molecular mechanism driving BFM mechanosensitivity is unknown. Here, we directly measure the kinetics of arrival and departure of the stator units in individual motors via analysis of high-resolution recordings of motor speed, while dynamically varying the load on the motor via external magnetic torque. The kinetic rates obtained, robust with respect to the details of the applied adsorption model, indicate that the lifetime of an assembled stator unit increases when a higher force is applied to its anchoring point in the cell wall. This provides strong evidence that a catch bond (a bond strengthened instead of weakened by force) drives mechanosensitivity of the flagellar motor complex. These results add the BFM to a short, but growing, list of systems demonstrating catch bonds, suggesting that this "molecular strategy" is a widespread mechanism to sense and respond to mechanical stress. We propose that force-enhanced stator adhesion allows the cell to adapt to a heterogeneous environmental viscosity and may ultimately play a role in surface-sensing during swarming and biofilm formation.


A modular platform for one-step assembly of multi-component membrane systems by fusion of charged proteoliposomes

Nature Communications Nature Publishing Group 7 (2016) 13025

RM Berry, R Ishmukhametov, AN Russell

An important goal in synthetic biology is the assembly of biomimetic cell-like structures, which combine multiple biological components in synthetic lipid vesicles. A key limiting assembly step is the incorporation of membrane proteins into the lipid bilayer of the vesicles. Here we present a simple method for delivery of membrane proteins into a lipid bilayer within 5 min. Fusogenic proteoliposomes, containing charged lipids and membrane proteins, fuse with oppositely charged bilayers, with no requirement for detergent or fusion-promoting proteins, and deliver large, fragile membrane protein complexes into the target bilayers. We demonstrate the feasibility of our method by assembling a minimal electron transport chain capable of adenosine triphosphate (ATP) synthesis, combining Escherichia coli F1Fo ATP-synthase and the primary proton pump bo3-oxidase, into synthetic lipid vesicles with sizes ranging from 100 nm to ∼10 μm. This provides a platform for the combination of multiple sets of membrane protein complexes into cell-like artificial structures.


Single-molecule imaging of electroporated dye-labelled CheY in live E. coli

Philosophical Transactions B Royal Society 371 (2016)

R Berry, D Di Paolo, J Armitage, O Afanzar

For the last two decades, the use of genetically fused Fluorescent Proteins has greatly contributed to the study of chemotactic signalling in E. coli including the activation of the response regulator protein CheY and its interaction with the flagellar motor. However, this approach suffers from a number of limitations, both biological and biophysical: for example, not all fusions are fully functional when fused to a bulky FP, which can have a similar molecular weight to its fused counterpart; they may interfere with the native interactions of the protein, and the chromophores of FPs have low brightness and photostability and fast photobleaching rates. A recently developed technique for the electroporation of fluorescently labelled proteins in live bacteria has enabled us to bypass these limitations and study the in vivobehaviour of CheY at the single molecule level. Here we show that purified CheY proteins labelled with organic dyes can be internalized into E. colicells in controllable concentrations and imaged with video fluorescence microscopy. The use of this approach is illustrated by showing single CheY molecules diffusing within cells and interacting with the sensory clusters and the flagellar motors in real time.


The limiting speed of the bacterial flagellar motor

Biophysical Journal Biophysical Society 111 (2016) 557–564-

RM Berry, JA Nirody, G Oster

Recent experiments on the bacterial flagellar motor have shown that the structure of this nanomachine, which drives locomotion in a wide range of bacterial species, is more dynamic than previously believed. Specifically, the number of active torque-generating complexes (stators) was shown to vary across applied loads. This finding brings under scrutiny the experimental evidence reporting that limiting (zero-torque) speed is independent of the number of active stators. Here, we propose that, contrary to previous assumptions, the maximum speed of the motor increases as additional stators are recruited. This result arises from our assumption that stators disengage from the motor for a significant portion of their mechanochemical cycles at low loads. We show that this assumption is consistent with current experimental evidence and consolidate our predictions with arguments that a processive motor must have a high duty ratio at high loads.


A Simple low-cost device enables four epi-illumination techniques on standard light microscopes

Scientific Reports Nature Publishing Group 6 (2016)

R Berry, R ishmukhametov, AN Russell, RJ Wheeler, AL Nord

Back-scattering darkfield (BSDF), epi-fluorescence (EF), interference reflection contrast (IRC), and darkfield surface reflection (DFSR) are advanced but expensive light microscopy techniques with limited availability. Here we show a simple optical design that combines these four techniques in a simple low-cost miniature epi-illuminator, which inserts into the differential interference-contrast (DIC) slider bay of a commercial microscope, without further additions required. We demonstrate with this device: 1) BSDF-based detection of Malarial parasites inside unstained human erythrocytes; 2) EF imaging with and without dichroic components, including detection of DAPI-stained Leishmania parasite without using excitation or emission filters; 3) RIC of black lipid membranes and other thin films, and 4) DFSR of patterned opaque and transparent surfaces. We believe that our design can expand the functionality of commercial bright field microscopes, provide easy field detection of parasites and be of interest to many users of light microscopy.


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

Nature structural & molecular biology 23 (2016) 197-203

MAB Baker, RMG Hynson, LA Ganuelas, NS Mohammadi, CW Liew, AA Rey, AP Duff, AE Whitten, CM Jeffries, NJ Delalez, YV Morimoto, D Stock, JP Armitage, AJ Turberfield, K Namba, RM Berry, LK Lee

Large protein complexes assemble spontaneously, yet their subunits do not prematurely form unwanted aggregates. This paradox is epitomized in the bacterial flagellar motor, a sophisticated rotary motor and sensory switch consisting of hundreds of subunits. Here we demonstrate that Escherichia coli FliG, one of the earliest-assembling flagellar motor proteins, forms ordered ring structures via domain-swap polymerization, which in other proteins has been associated with uncontrolled and deleterious protein aggregation. Solution structural data, in combination with in vivo biochemical cross-linking experiments and evolutionary covariance analysis, revealed that FliG exists predominantly as a monomer in solution but only as domain-swapped polymers in assembled flagellar motors. We propose a general structural and thermodynamic model for self-assembly, in which a structural template controls assembly and shapes polymer formation into rings.


Mutations targeting the plug-domain of the Shewanella oneidensis proton-driven stator allow swimming at increased viscosity and under anaerobic conditions

Molecular Microbiology John Wiley & Sons Ltd 102 (2016) 925–938-

KM Thormann, S Brenzinger, L Dewenter, NJ Delalez, O Leicht, V Berndt, RM Berry, M Thanbichler, J Armitage, B Maier

Shewanella oneidensis MR-1 possesses two different stator units to drive flagellar rotation, the Na+-dependent PomAB stator and the H+-driven MotAB stator, the latter possibly acquired by lateral gene transfer. Although either stator can independently drive swimming through liquid, MotAB-driven motors cannot support efficient motility in structured environments or swimming under anaerobic conditions. Using ΔpomAB cells we isolated spontaneous mutants able to move through soft agar. We show that a mutation that alters the structure of the plug domain in MotB affects motor functions and allows cells to swim through media of increased viscosity and under anaerobic conditions. The number and exchange rates of the mutant stator around the rotor were not significantly different from wild-type stators, suggesting that the number of stators engaged is not the cause of increased swimming efficiency. The swimming speeds of planktonic mutant MotAB-driven cells was reduced, and overexpression of some of these stators caused reduced growth rates, implying that mutant stators not engaged with the rotor allow some proton leakage. The results suggest that the mutations in the MotB plug domain alter the proton interactions with the stator ion channel in a way that both increases torque output and allows swimming at decreased pmf values. This article is protected by copyright. All rights reserved.


Transient pauses of the bacterial flagellar motor at low load

NEW JOURNAL OF PHYSICS 18 (2016) ARTN 115002

AL Nord, F Pedaci, RM Berry


Rotational Measurements and Manipulations of the Bacterial Flagellar Motor

BIOPHYSICAL JOURNAL 110 (2016) 198A-198A

AL Nord, RM Berry, F Pedaci


Single-molecule imaging of electroporated dye-labelled CheY in live Escherichia coli

Philosophical Transactions B: Biological Sciences Royal Society 371 (2016) 20150492-

R Berry, D Di Paolo, O Afanzar, J Armitage

For the past two decades, the use of genetically fused fluorescent proteins (FPs) has greatly contributed to the study of chemotactic signalling in Escherichia coli including the activation of the response regulator protein CheY and its interaction with the flagellar motor. However, this approach suffers from a number of limitations, both biological and biophysical: for example, not all fusions are fully functional when fused to a bulky FP, which can have a similar molecular weight to its fused counterpart; they may interfere with the native interactions of the protein and the chromophores of FPs have low brightness and photostability and fast photobleaching rates. A recently developed technique for the electroporation of fluorescently labelled proteins in live bacteria has enabled us to bypass these limitations and study the in vivo behaviour of CheY at the single-molecule level. Here we show that purified CheY proteins labelled with organic dyes can be internalized into E. coli cells in controllable concentrations and imaged with video fluorescence microscopy. The use of this approach is illustrated by showing single CheY molecules diffusing within cells and interacting with the sensory clusters and the flagellar motors in real time.


A simple low-cost device enables four epi-illumination techniques on standard light microscopes

Scientific Reports Nature Publishing Group 6 (2016) 20729

R Ishmukhametov, AN Russell, RJ Wheeler, AL Nord, RM Berry

Back-scattering darkfield (BSDF), epi-fluorescence (EF), interference reflection contrast (IRC), and darkfield surface reflection (DFSR) are advanced but expensive light microscopy techniques with limited availability. Here we show a simple optical design that combines these four techniques in a simple low-cost miniature epi-illuminator, which inserts into the differential interference-contrast (DIC) slider bay of a commercial microscope, without further additions required. We demonstrate with this device: 1) BSDF-based detection of Malarial parasites inside unstained human erythrocytes; 2) EF imaging with and without dichroic components, including detection of DAPI-stained Leishmania parasite without using excitation or emission filters; 3) RIC of black lipid membranes and other thin films, and 4) DFSR of patterned opaque and transparent surfaces. We believe that our design can expand the functionality of commercial bright field microscopes, provide easy field detection of parasites and be of interest to many users of light microscopy.


Mechanics of torque generation in the bacterial flagellar motor

Proceedings of the National Academy of Sciences National Academy of Sciences 112 (2015) E4381-E4389

KK Mandadapu, JA Nirody, R Berry, G Oster

The bacterial flagellar motor (BFM) is responsible for driving bacterial locomotion and chemotaxis, fundamental processes in pathogenesis and biofilm formation. In the BFM, torque is generated at the interface between transmembrane proteins (stators) and a rotor. It is well established that the passage of ions down a transmembrane gradient through the stator complex provides the energy for torque generation. However, the physics involved in this energy conversion remain poorly understood. Here we propose a mechanically specific model for torque generation in the BFM. In particular, we identify roles for two fundamental forces involved in torque generation: electrostatic and steric. We propose that electrostatic forces serve to position the stator, whereas steric forces comprise the actual “power stroke.” Specifically, we propose that ion-induced conformational changes about a proline “hinge” residue in a stator α-helix are directly responsible for generating the power stroke. Our model predictions fit well with recent experiments on a single-stator motor. The proposed model provides a mechanical explanation for several fundamental properties of the flagellar motor, including torque–speed and speed–ion motive force relationships, backstepping, variation in step sizes, and the effects of key mutations in the stator.

Pages