Ashley L. Nord

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

The rotary motor F1-ATPase from the thermophilic Bacillus PS3 (TF1) is one of the best-studied of all molecular machines. F1-ATPase is the part of the enzyme F1FO-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 F1-ATPase from Escherichia coli (EF1) is governed by the same mechanism as TF1 under laboratory conditions. Using optical microscopy to measure rotation of a variety of marker particles attached to the γ-subunit of single surface-bound EF1 molecules, we characterized the ATP-binding, catalytic and inhibited states of EF1. We also show that the ATP-binding and catalytic states are separated by 35±3°. At room temperature, chemical processes occur faster in EF1 than in TF1, 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.

Catch bond drives stator mechanosensitivity in the bacterial flagellar motor

Significance The bacterial flagellar motor (BFM) is the rotary motor powering swimming of many motile bacteria. Many of the components of this molecular machine are dynamic, a property which allows the cell to optimize its behavior in accordance with the surrounding environment. A prime example is the stator unit, a membrane-bound ion channel that is responsible for applying torque to the rotor. The stator units are mechanosensitive, with the number of engaged units dependent on the viscous load on the motor. We measure the kinetics of the stators as a function of the viscous load and find that the mechanosensitivity of the BFM is governed by a catch bond: a counterintuitive type of bond that becomes stronger under force. 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 Simple low-cost device enables four epi-illumination techniques on standard light microscopes.

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.

Speed of the bacterial flagellar motor near zero load depends on the number of stator units

Significance The bacterial flagellar motor is a rotary molecular motor responsible for swimming, swarming, and chemotaxis in many species of bacteria. It generates torque by interactions between a rotor 50 nm in diameter and multiple stator units. We overturn the prevailing understanding of how stator units interact with each other by showing that motor speed is dependent on the number of stator units even at high speed near zero torque. We describe a method to measure torque and speed that uses synthetic hybrid stators driven by different ion types and show that, with simple rescaling, a single torque–speed curve describes the motor over widely varying values of the membrane voltage, driving ion type and ionic chemical potential gradient. The bacterial flagellar motor (BFM) rotates hundreds of times per second to propel bacteria driven by an electrochemical ion gradient. The motor consists of a rotor 50 nm in diameter surrounded by up to 11 ion-conducting stator units, which exchange between motors and a membrane-bound pool. Measurements of the torque–speed relationship guide the development of models of the motor mechanism. In contrast to previous reports that speed near zero torque is independent of the number of stator units, we observe multiple speeds that we attribute to different numbers of units near zero torque in both Na+- and H+-driven motors. We measure the full torque–speed relationship of one and two H+ units in Escherichia coli by selecting the number of H+ units and controlling the number of Na+ units in hybrid motors. These experiments confirm that speed near zero torque in H+-driven motors increases with the stator number. We also measured 75 torque–speed curves for Na+-driven chimeric motors at different ion-motive force and stator number. Torque and speed were proportional to ion-motive force and number of stator units at all loads, allowing all 77 measured torque–speed curves to be collapsed onto a single curve by simple rescaling.

Comparison between single-molecule and X-ray crystallography data on yeast F 1 -ATPase

Single molecule studies in recent decades have elucidated the full chemo-mechanical cycle of F1-ATPase, mostly based on F1 from thermophilic bacteria. In contrast, high-resolution crystal structures are only available for mitochondrial F1. Here we present high resolution single molecule rotational data on F1 from Saccharomyces cerevisiae, obtained using new high throughput detection and analysis tools. Rotational data are presented for the wild type mitochondrial enzyme, a “liver” isoform, and six mutant forms of yeast F1 that have previously been demonstrated to be less efficient or partially uncoupled. The wild-type and “liver” isoforms show the same qualitative features as F1 from Escherichia coli and thermophilic bacteria. The analysis of the mutant forms revealed a delay at the catalytic dwell and associated decrease in Vmax, with magnitudes consistent with the level of disruption seen in the crystal structures. At least one of the mutant forms shows a previously un-observed dwell at the ATP binding angle, potentially attributable to slowed release of ADP. We discuss the correlation between crystal structures and single molecule results.

Transient pauses of the bacterial flagellar motor at low load

The bacterial flagellar motor (BFM) is the molecular machine responsible for the swimming and chemotaxis of many species of motile bacteria. The BFM is bidirectional, and changes in the rotation direction of the motor are essential for chemotaxis. It has previously been observed that many species of bacteria also demonstrate brief pauses in rotation, though the underlying cause of such events remains poorly understood. We examine the rotation of Escherichia coli under low mechanical load with high spatial and temporal resolution. We observe and characterize transient pauses in rotation in a strain which lacks a functional chemosensory network, showing that such events are a phenomenon separate from a change in rotational direction. Rotating at low load, the BFM of E. coli exhibits about 10 pauses per second, lasting on average 5 ms, during which time the rotor diffuses with net forwards rotation. Replacing the wild type stators with Na+ chimera stators has no substantial effect on the pausing. We discuss possible causes of such events, which are likely a product of a transient change in either the stator complex or the rotor.

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

Nature Reviews Microbiology 10, 583–588 (2012) In the original article, the order and citations for references 1 and 2 was incorrect. In the section Sprint, the reference citation should have been as follows: “A chant erupts from the eukaryotic crowd: “Kill the winner! Kill the winner!” (REF. 2.)”. In the reference list, references 1 and 2 were listed in the wrong order; this has now been corrected as listed below. We apologize to the authors and to readers for this error and for any confusion caused.