Publications


Fluorescence measurement of intracellular sodium concentration in single Escherichia coli cells.

Biophys J 90 (2006) 357-365

C-J Lo, MC Leake, RM Berry

The energy-transducing cytoplasmic membrane of bacteria contains pumps and antiports maintaining the membrane potential and ion gradients. We have developed a method for rapid, single-cell measurement of the internal sodium concentration ([Na(+)](in)) in Escherichia coli using the sodium ion fluorescence indicator, Sodium Green. The bacterial flagellar motor is a molecular machine that couples the transmembrane flow of ions, either protons (H(+)) or sodium ions (Na(+)), to flagellar rotation. We used an E. coli strain containing a chimeric flagellar motor with H(+)- and Na(+)-driven components that functions as a sodium motor. Changing external sodium concentration ([Na(+)](ex)) in the range 1-85 mM resulted in changes in [Na(+)](in) between 5-14 mM, indicating a partial homeostasis of internal sodium concentration. There were significant intercell variations in the relationship between [Na(+)](in) and [Na(+)](ex), and the internal sodium concentration in cells not expressing chimeric flagellar motors was 2-3 times lower, indicating that the sodium flux through these motors is a significant fraction of the total sodium flux into the cell.


Torque-speed relationship of the bacterial flagellar motor

Proceedings of the National Academy of Sciences of the USA 103 (2006) 1260-1265

RM Berry, Bai F, Oster G, Xing J


Controlled delivery of membrane proteins to artificial lipid bilayers by nystatin-ergosterol modulated vesicle fusion.

IEE Proc Nanobiotechnol 153 (2006) 21-30

MRR de Planque, MRR de Planque, GP Mendes, M Zagnoni, ME Sandison, KH Fisher, RM Berry, A Watts, H Morgan

The study of ion channels and other membrane proteins and their potential use as biosensors and drug screening targets require their reconstitution in an artificial membrane. These applications would greatly benefit from microfabricated devices in which stable artificial lipid bilayers can be rapidly and reliably formed. However, the amount of protein delivered to the bilayer must be carefully controlled. A vesicle fusion technique is investigated where composite ion channels of the polyene antibiotic nystatin and the sterol ergosterol are employed to render protein-carrying vesicles fusogenic. After fusion with an ergosterol-free artificial bilayer, the nystatin-ergosterol channels do not dissociate immediately and thus cause a transient current signal that marks the vesicle fusion event. Experimental pitfalls of this method were identified, the influence of the nystatin and ergosterol concentration on the fusion rate and the shape of the fusion event marker was explored, and the number of different lipid species was reduced. Under these conditions, the -amyloid peptide could be delivered in a controlled manner to a standard planar bilayer. Additionally, electrical recordings were obtained of vesicles fusing with a planar lipid bilayer in a microfabricated device, demonstrating the suitability of nystatin-ergosterol modulated vesicle fusion for protein delivery within microsystems.


Artificial bilayer lipid membranes (BLMs) on-chip for single molecule sensing

Proceedings of SPIE - The International Society for Optical Engineering 5838 (2005) 252-257

ME Sandison, D Malleo, D Holmes, R Berry, H Morgan

A polymer microfluidic device for the formation of artificial bilayer lipid membranes (BLMs) on-chip is described. The device is fabricated from thin, transparent films of poly(methyl methacrylate), allowing for optical monitoring of the BLM. In addition, detection of single fluorescently-labeled lipid molecules using conventional epifluorescence microscopy is described.


ATP synthesis: the world's smallest wind-up toy.

Curr Biol 15 (2005) R385-R387

RM Berry

ATP synthase contains two rotary motors coupled back-to-back: the protonmotive force-driven motor F0 pushes the ATP-driven motor F1 in reverse, causing it to synthesize ATP. Half of this process has now been reproduced in vitro, using tiny magnets instead of F0 to drive the reverse rotation of a single F1 molecule.


Intracellular sodium concentration of chimera Escherichia coli

FEBS J 272 (2005) 345-346

CJ Lo, MC Leake, RM Berry


Chemistry: Rapid chiral assembly of rigid DNA building blocks for molecular nanofabrication

Science 310 (2005) 1661-1665

RP Goodman, IAT Schaap, CF Tardin, CM Erben, RM Berry, CF Schmidt, AJ Turberfield

Practical components for three-dimensional molecular nanofabrication must be simple to produce, stereopure, rigid, and adaptable. We report a family of DNA tetrahedra, less than 10 nanometers on a side, that can self-assemble in seconds with near-quantitative yield of one diastereomer. They can be connected by programmable DNA linkers. Their triangulated architecture confers structural stability; by compressing a DNA tetrahedron with an atomic force microscope, we have measured the axial compressibility of DNA and observed the buckling of the double helix under high loads.


Direct observation of steps in rotation of the bacterial flagellar motor

Nature 437 (2005) 916-919

RM Berry, Leake MC, Rowe AD, Sowa Y


Rapid chiral assembly of rigid DNA building blocks for molecular nanofabrication.

Science 310 (2005) 1661-1665

RP Goodman, IAT Schaap, CF Tardin, CM Erben, RM Berry, CF Schmidt, AJ Turberfield

Practical components for three-dimensional molecular nanofabrication must be simple to produce, stereopure, rigid, and adaptable. We report a family of DNA tetrahedra, less than 10 nanometers on a side, that can self-assemble in seconds with near-quantitative yield of one diastereomer. They can be connected by programmable DNA linkers. Their triangulated architecture confers structural stability; by compressing a DNA tetrahedron with an atomic force microscope, we have measured the axial compressibility of DNA and observed the buckling of the double helix under high loads.


Steps in slow flagellar motor rotation

BIOPHYS J 88 (2005) 504A-504A

AD Rowe, Y Sowa, MC Leake, T Yakushi, M Homma, A Ishijima, RM Berry


E. Coli in motion

Physics Today 58 (2005) 64-65

HC Berg, RM Berry


The bacterial flagellar motor

NATO SCI SER II MATH 160 (2004) 145-164

RM Berry


The single-step synthesis of a DNA tetrahedron.

Chem Commun (Camb) (2004) 1372-1373

RP Goodman, RM Berry, AJ Turberfield

A tetrahedral nanostructure whose edges are DNA double helices self-assembles spontaneously when four appropriately designed oligonucleotides are annealed together in solution; the ease of synthesis, rigidity, and adaptability of this construct make it a promising candidate as a cage for other large molecules and as a building block for more complicated nanostructures.


Rapid rotation of micron and submicron dielectric particles measured using optical tweezers

Journal of Modern Optics 50 (2003) 1539-1554

AD Rowe, MC Leake, H Morgan, RM Berry

We demonstrate the use of a laser trap (‘optical tweezers’) and back-focal-plane position detector to measure rapid rotation in aqueous solution of single particles with sizes in the vicinity of 1 μm. Two types of rotation were measured: electrorotation of polystyrene microspheres and rotation of the flagellar motor of the bacterium Vibrio alginolyticus. In both cases, speeds in excess of 1000 Hz (rev s−1) were measured. Polystyrene beads of diameter about 1 μm labelled with smaller beads were held at the centre of a microelectrode array by the optical tweezers. Electrorotation of the labelled beads was induced by applying a rotating electric field to the solution using microelectrodes. Electrorotation spectra were obtained by varying the frequency of the applied field and analysed to obtain the surface conductance of the beads. Single cells of V. alginolyticus were trapped and rotation of the polar sodium-driven flagellar motor was measured. Cells rotated more rapidly in media containing higher concentrations of Na+, and photodamage caused by the trap was considerably less when the suspending medium did not contain oxygen. The technique allows single-speed measurements to be made in less than a second and separate particles can be measured at a rate of several per minute. © 2003 Taylor & Francis Group, LLC.


Torque-speed relationship of the flagellar rotary motor of Rhodobacter using an electrorotation technique

BIOPHYSICAL JOURNAL 82 (2002) 401A-402A

MC Leake, RM Berry


Response kinetics of tethered Rhodobacter sphaeroides to changes in light intensity.

Biophys J 78 (2000) 1207-1215

RM Berry, JP Armitage

Rhodobacter sphaeroides can swim toward a wide range of attractants (a process known as taxis), propelled by a single rotating flagellum. The reversals of motor direction that cause tumbles in Eschericia coli taxis are replaced by brief motor stops, and taxis is controlled by a complex sensory system with multiple homologues of the E. coli sensory proteins. We tethered photosynthetically grown cells of R. sphaeroides by their flagella and measured the response of the flagellar motor to changes in light intensity. The unstimulated bias (probability of not being stopped) was significantly larger than the bias of tethered E. coli but similar to the probability of not tumbling in swimming E. coli. Otherwise, the step and impulse responses were the same as those of tethered E. coli to chemical attractants. This indicates that the single motor and multiple sensory signaling pathways in R. sphaeroides generate the same swimming response as several motors and a single pathway in E. coli, and that the response of the single motor is directly observable in the swimming pattern. Photo-responses were larger in the presence of cyanide or the uncoupler carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP), consistent with the photo-response being detected via changes in the rate of electron transport.


Response kinetics of tethered Rhodobacter sphaeroides to changes in light intensity

Biophysical Journal 78 (2000) 1207-1215

RM Berry, JP Armitage

Rhodobacter sphaeroides can swim toward a wide range of attractants (a process known as taxis), propelled by a single rotating flagellum. The reversals of motor direction that cause tumbles in Eschericia coli taxis are replaced by brief motor stops, and taxis is controlled by a complex sensory system with multiple homologues of the E. coli sensory proteins. We tethered photosynthetically grown cells of R. sphaeroides by their flagella and measured the response of the flagellar motor to changes in light intensity. The unstimulated bias (probability of not being stopped) was significantly larger than the bias of tethered E. coli but similar to the probability of not tumbling in swimming E. coli. Otherwise, the step and impulse responses were the same as those of tethered E. coli to chemical attractants. This indicates that the single motor and multiple sensory signaling pathways in R. sphaeroides generate the same swimming response as several motors and a single pathway in E. coli, and that the response of the single motor is directly observable in the swimming pattern. Photo-responses were larger in the presence of cyanide or the uncoupler carbonyl cyanide 4- trifluoromethoxyphenylhydrazone (FCCP), consistent with the photo-response being detected via changes in the rate of electron transport.


Torque-generating units of the flagellar motor of Escherichia coli have a high duty ratio.

Nature 403 (2000) 444-447

WS Ryu, RM Berry, HC Berg

Rotation of the bacterial flagellar motor is driven by an ensemble of torque-generating units containing the proteins MotA and MotB. Here, by inducing expression of MotA in motA- cells under conditions of low viscous load, we show that the limiting speed of the motor is independent of the number of units: at vanishing load, one unit turns the motor as rapidly as many. This result indicates that each unit may remain attached to the rotor for most of its mechanochemical cycle, that is, that it has a high duty ratio. Thus, torque generators behave more like kinesin, the protein that moves vesicles along microtubules, than myosin, the protein that powers muscle. However, their translation rates, stepping frequencies and power outputs are much higher, being greater than 30 microm s(-1), 12 kHz and 1.5 x 10(5) pN nm s(-1), respectively.


Theories of rotary motors.

Philosophical transactions of the Royal Society of London. Series B, Biological sciences 355 (2000) 503-509

RM Berry

The bacterial flagellar motor and the ATP-hydrolysing F1 portion of the F1Fo-ATPase are known to be rotary motors, and it seems highly probable that the H+-translocating Fo portion rotates too. The energy source in the case of Fo and the flagellar motor is the flow of ions, either H+ (protons) or Na+, down an electrochemical gradient across a membrane. The fact that ions flow in a particular direction through a well-defined structure in these motors invites the possibility of a type of mechanism based on geometric constraints between the rotor position and the paths of ions flowing through the motor. The two best-studied examples of such a mechanism are the 'turnstile' model of Khan and Berg and the 'proton turbine' model of Läuger or Berry. Models such as these are typically represented by a small number of kinetic states and certain allowed transitions between them. This allows the calculation of predictions of motor behaviour and establishes a dialogue between models and experimental results. In the near future structural data and observations of single-molecule events should help to determine the nature of the mechanism of rotary motors, while motor models must be developed that can adequately explain the measured relationships between torque and speed in the flagellar motor.


Torque generated by the flagellar motor of Escherichia coli while driven backward.

Biophysical journal 76 (1999) 580-587

RM Berry, HC Berg

The technique of electrorotation was used to apply torque to cells of the bacterium Escherichia coli tethered to glass coverslips by single flagella. Cells were made to rotate backward, that is, in the direction opposite to the rotation driven by the flagellar motor itself. The torque generated by the motor under these conditions was estimated using an analysis that explicitly considers the angular dependence of both the viscous drag coefficient of the cell and the torque produced by electrorotation. Motor torque varied approximately linearly with speed up to over 100 Hz in either direction, placing constraints on mechanisms for torque generation in which rates of proton transfer for backward rotation are limiting. These results, interpreted in the context of a simple three-state kinetic model, suggest that the rate-limiting step in the torque-generating cycle is a powerstroke in which motor rotation and dissipation of the energy available from proton transit occur synchronously.

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