Teuta Pilizota

A molecular brake, not a clutch, stops the Rhodobacter sphaeroides flagellar motor

Many bacterial species swim by employing ion-driven molecular motors that power the rotation of helical filaments. Signals are transmitted to the motor from the external environment via the chemotaxis pathway. In bidirectional motors, the binding of phosphorylated CheY (CheY-P) to the motor is presumed to instigate conformational changes that result in a different rotor-stator interface, resulting in rotation in the alternative direction. Controlling when this switch occurs enables bacteria to accumulate in areas favorable for their survival. Unlike most species that swim with bidirectional motors, Rhodobacter sphaeroides employs a single stop-start flagellar motor. Here, we asked, how does the binding of CheY-P stop the motor in R. sphaeroides—using a clutch or a brake? By applying external force with viscous flow or optical tweezers, we show that the R. sphaeroides motor is stopped using a brake. The motor stops at 27–28 discrete angles, locked in place by a relatively high torque, approximately 2–3 times its stall torque.

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.

Fast, Multiphase Volume Adaptation to Hyperosmotic Shock by Escherichia coli

All living cells employ an array of different mechanisms to help them survive changes in extra cellular osmotic pressure. The difference in the concentration of chemicals in a bacteriums cytoplasm and the external environment generates an osmotic pressure that inflates the cell. It is thought that the bacterium Escherichia coli use a number of interconnected systems to adapt to changes in external pressure, allowing them to maintain turgor and live in surroundings that range more than two-hundred-fold in external osmolality. Here, we use fluorescence imaging to make the first measurements of cell volume changes over time during hyperosmotic shock and subsequent adaptation on a single cell level in vivo with a time resolution on the order of seconds. We directly observe two previously unseen phases of the cytoplasmic water efflux upon hyperosmotic shock. Furthermore, we monitor cell volume changes during the post-shock recovery and observe a two-phase response that depends on the shock magnitude. The initial phase of recovery is fast, on the order of 15–20 min and shows little cell-to-cell variation. For large sucrose shocks, a secondary phase that lasts several hours adds to the recovery. We find that cells are able to recover fully from shocks as high as 1 Osmol/kg using existing systems, but that for larger shocks, protein synthesis is required for full recovery.

Mechanism and kinetics of a sodium-driven bacterial flagellar motor

Significance Using new experimental methods, we measure the mechanical output of the bacterial flagellar motor, the rotary molecular machine that propels swimming bacteria, while varying both electrical and chemical components of the ion-motive force that drives it. We find that each independent torque-generating stator in the motor passes 37 ± 2 ions per revolution, at odds with previous indications of 26 or 52 ions. Fitting our data to theoretical models reveals the kinetics of the motor mechanism. Our thorough search of the multidimensional parameter space of generalized motor models, guided by experimental data, is an approach that may be widely applicable. The bacterial flagellar motor is a large rotary molecular machine that propels swimming bacteria, powered by a transmembrane electrochemical potential difference. It consists of an ∼50-nm rotor and up to ∼10 independent stators anchored to the cell wall. We measured torque–speed relationships of single-stator motors under 25 different combinations of electrical and chemical potential. All 25 torque–speed curves had the same concave-down shape as fully energized wild-type motors, and each stator passes at least 37 ± 2 ions per revolution. We used the results to explore the 25-dimensional parameter space of generalized kinetic models for the motor mechanism, finding 830 parameter sets consistent with the data. Analysis of these sets showed that the motor mechanism has a “powerstroke” in either ion binding or transit; ion transit is channel-like rather than carrier-like; and the rate-limiting step in the motor cycle is ion binding at low concentration, ion transit, or release at high concentration.

General calibration of microbial growth in microplate readers

Optical density (OD) measurements of microbial growth are one of the most common techniques used in microbiology, with applications ranging from studies of antibiotic efficacy to investigations of growth under different nutritional or stress environments, to characterization of different mutant strains, including those harbouring synthetic circuits. OD measurements are performed under the assumption that the OD value obtained is proportional to the cell number, i.e. the concentration of the sample. However, the assumption holds true in a limited range of conditions, and calibration techniques that determine that range are currently missing. Here we present a set of calibration procedures and considerations that are necessary to successfully estimate the cell concentration from OD measurements.

Origins of Escherichia coli Growth Rate and Cell Shape Changes at High External Osmolality

In Escherichia coli, a sudden increase in external concentration causes a pressure drop across the cell envelope, followed by an active recovery. After recovery, and if the external osmolality remains high, cells have been shown to grow more slowly, smaller, and at reduced turgor pressure. Despite the fact that the active recovery is a key stress response, the nature of these changes and how they relate to each other is not understood. Here, we use fluorescence imaging of single cells during hyperosmotic shocks, combined with custom made microfluidic devices, to show that cells fully recover their volume to the initial, preshock value and continue to grow at a slower rate immediately after the recovery. We show that the cell envelope material properties do not change after hyperosmotic shock, and that cell shape recovers along with cell volume. Taken together, these observations indicate that the turgor pressure recovers to its initial value so that reduced turgor is not responsible for the reduced growth rate observed immediately after recovery. To determine the point at which the reduction in cell size and turgor pressure occurs after shock, we measured the volume of E. coli cells at different stages of growth in bulk cultures. We show that cell volume reaches the same maximal level irrespective of the osmolality of the media. Based on these measurements, we propose that turgor pressure is used as a feedback variable for osmoregulatory pumps instead of being directly responsible for the reduction in growth rates. Reestablishment of turgor to its initial value might ensure correct attachment of the inner membrane and cell wall needed for cell wall biosynthesis.

Dynamics of Escherichia coli’s passive response to a sudden decrease in external osmolarity

Significance Mechanosensation is central to life. Bacteria, like the majority of walled cells, live and grow under significant osmotic pressure. By relying on mechanosensitive regulation, bacteria can adapt to dramatic changes in osmotic pressure. Studying such mechanical sensing and control is critical for understanding bacterial survival in a complex host and natural environment. Here, we investigate the fundamental design principles of Escherichia coli’s passive mechanosensitive response to osmotic downshocks by implementing single-cell high-resolution imaging. We explain the observed cell volume changes by modeling flux of water and solutes across the cell membrane. A better characterization of bacterial mechanosensitive response can help us map their reaction to environmental threats. For most cells, a sudden decrease in external osmolarity results in fast water influx that can burst the cell. To survive, cells rely on the passive response of mechanosensitive channels, which open under increased membrane tension and allow the release of cytoplasmic solutes and water. Although the gating and the molecular structure of mechanosensitive channels found in Escherichia coli have been extensively studied, the overall dynamics of the whole cellular response remain poorly understood. Here, we characterize E. coli’s passive response to a sudden hypoosmotic shock (downshock) on a single-cell level. We show that initial fast volume expansion is followed by a slow volume recovery that can end below the initial value. Similar response patterns were observed at downshocks of a wide range of magnitudes. Although wild-type cells adapted to osmotic downshocks and resumed growing, cells of a double-mutant (ΔmscL,ΔmscS) strain expanded, but failed to fully recover, often lysing or not resuming growth at high osmotic downshocks. We propose a theoretical model to explain our observations by simulating mechanosensitive channels opening, and subsequent solute efflux and water flux. The model illustrates how solute efflux, driven by mechanical pressure and solute chemical potential, competes with water influx to reduce cellular osmotic pressure and allow volume recovery. Our work highlights the vital role of mechanosensation in bacterial survival.

Painting with light-powered bacteria

External control of the swimming speed of ‘active particles’ can be used to self assemble designer structures in situ on the μm to mm scale. We demonstrate such reconfigurable templated active self assembly in a fluid environment using light powered strains of Escherichia coli. The physics and biology controlling the sharpness and formation speed of patterns is investigated using a bespoke fast-responding strain. Microand nano-fabrication can revolutionise many areas of technology, including personalised medicine. There are two conceptually distinct ways to construct structures on the 10 nm to 10 μm scale: lithography, which uses ‘scalpels’ such as chemical etching or electron beams, or self assembly[1], in which microscopic ‘Lego components’ move themselves into position. Both equilibrium phase transitions (e.g. crystallization) and nonequilibrium processes are exploited for self assembly. In either case, external templates can be used to direct the process, with reconfigurable templates offering programmability. Self assembly was originally inspired by chemistry and biology, where the components are individual molecules. Increasingly, colloidal building blocks are used, with bespoke particle shape, size and interaction, e.g. ‘patchy particles’ with heterogeneous surface chemistry[2]. Active, or self-propelled, colloids open up further opportunities. We show how to assemble structures on the μm to mm scale that are reconfigurable in real time using Escherichia coli bacteria – ‘living active colloids’ – that swim only when illuminated[3]. The process is directed by a smart, or programmable, external template applied by a spatial light modulator. Active colloids, or self-propelled micro-swimmers, are attracting significant recent attention[4] as ‘active matter’[5, 6]. They violate time-reversal symmetry[7], and may be used, e.g., to transport colloidal ‘cargos’[8]. For both fundamental physics and applications, external control of swimming, e.g. using particles with lightactivated self-propulsion[9, 10, 11, 12], opens up many new possibilities. Thus, e.g., light-activated motile bacteria can be used to actuate and control micro-machinery [13]. ∗Electronic address: j.arlt@ed.ac.uk; Corresponding author 1 ar X iv :1 71 0. 08 18 8v 1 [ co nd -m at .s of t] 2 3 O ct 2 01 7 The self assembly of micro-swimmers into clusters of tens of particles has already been demonstrated[14, 15, 12]. Recent simulations[16] suggest that the patterned illumination of light-activated swimmers can be used for the templated self assembly[1] of designer structures comprising 10-10 particles. Real-time reconfiguration of the light field then allows smart templated active self assembly (STASA), which we here implement for the first time using light-controlled motile bacteria. E. coli bacteria[17] (cell body ≈ 2 μm×1 μm) swim by turning ≈ 7-10 μm long helical flagella using membraneembedded rotary motors powered by a protonmotive force (PMF) that arises from active pumping of H to the extracellular medium[18]. Unlike all synthetic active colloids to date and most bacteria, E. coli can generate PMF in nutrient-free motility buffer[19] by utilising internal resources and oxygen (O2) to produce energetic electron pairs. These release their energy stepwise along an electrochemical potential ladder of respiratory enzymes located in the inner cell membrane, generating a PMF of ≈ −150 mV. The electron pair ultimately passes to and reduces O2 to water. Thus, with no O2, PMF = 0 and swimming ceases[17]. If cells under anaerobic conditions can express proteorhodopsin (PR)[3], a green-photon-driven proton pump[20], then they will swim only when suitably illuminated: these are living analogues of synthetic light-activated colloidal swimmers.[9, 10, 11, 12] We show below that the speed with which such cells respond to changes in illumination is crucial for successful bacterial STASA. For this work, we constructed a PR-bearing mutant (AD10) that stops much faster when illumination ceases than previously-reported[3, 21], by deleting the unc gene cluster[22] encoding the F1FoATPase membrane protein complex (see SI §1.1), so that these enzymes cannot act in reverse in darkness to continue to export protons and sustain a PMF[23]. We suspended cells in phosphate motility buffer at optical density OD . 8 (cell-body volume fraction ≈ 1.1%)[17] and sealed 2 μL into 20 μm high flat capillaries, where cells swim in two dimensions but have enough room to ‘overtake’ each other in all three spatial dimensions. Differential dynamic microscopy (DDM)[24] returned an averaged speed v̄ ≈ 30 μm s−1 and β ≈ 20% of non-motile organisms at OD=1 under fully-oxygenated conditions. (Note that ‘non-motile’ = cells that can never swim; ‘stationary’ = non-swimming cells capable of motility when illuminated.) Motile cells were allowed to swim until O2 was depleted and v̄ dropped abruptly to zero after a few minutes[17] (see SI §1.2 and Fig. S1(a)). After these cells were left in the dark for ≈ 10 min, green illumination was turned on (510 – 560 nm, intensity I ≈ 5 mW cm−2 at the sample). The stationary cells accelerated uniformly before saturating, Fig. 1. Fitting the data to v̄(t) = v̄satt/(t+τon) gives v̄sat = 9.5 μm s−1, τon = 30 s. When illumination ceased, v̄ dropped within τoff . 1 s, but never quite to zero – it is unclear why a few cells (< 1%) continued to swim. v̄sat increased with I, Fig. 1 (inset), up to . 27 μm s−1. {v̄, β, vsat, τon} changed over hours as cells aged. The discharging of the PMF through the membrane (capacitance C & 10−14 F) and rotary motors (total resistance R . 10 Ω) upon cessation of illumination should take RC ∼ 1 s (see SI §1.3 for details), which explains the observed τoff . Consistent with this interpretation, τoff is approximately independent of the starting speed of decelerating cells (see SI §1.2 and Fig. S2(a)). The observed τon ≈ 30 s is likely controlled by the rate constant[25] for stator units to come on and off motors, kstator ≈ 0.04 s−1 ∼ τ−1 on . In sustained darkness, motors disassemble in PR-bearing E. coli, and full ‘motor resurrection’ upon illumination takes[21] ∼ 200 s, in agreementSelf-assembly is a promising route for micro- and nano-fabrication with potential to revolutionise many areas of technology, including personalised medicine. Here we demonstrate that external control of the swimming speed of microswimmers can be used to self assemble reconfigurable designer structures in situ. We implement such ‘smart templated active self assembly’ in a fluid environment by using spatially patterned light fields to control photon-powered strains of motile Escherichia coli bacteria. The physics and biology governing the sharpness and formation speed of patterns is investigated using a bespoke strain designed to respond quickly to changes in light intensity. Our protocol provides a distinct paradigm for self-assembly of structures on the 10 μm to mm scale.The ability to generate microscale patterns and control microswimmers may be useful for engineering smart materials. Here Arlt et al. use genetically modified bacteria with fast response to changes in light intensity to produce light-induced patterns.

Inferring time derivatives, including cell growth rates, using Gaussian processes

Often the time derivative of a measured variable is of as much interest as the variable itself. For a growing population of biological cells, for example, the populations growth rate is typically more important than its size. Here we introduce a non-parametric method to infer first and second time derivatives as a function of time from time-series data. Our approach is based on Gaussian processes and applies to a wide range of data. In tests, the method is at least as accurate as others, but has several advantages: it estimates errors both in the inference and in any summary statistics, such as lag times, and allows interpolation with the corresponding error estimation. As illustrations, we infer growth rates of microbial cells, the rate of assembly of an amyloid fibril and both the speed and acceleration of two separating spindle pole bodies. Our algorithm should thus be broadly applicable.

Single-Molecule Studies of Rotary Molecular Motors

Rotary molecular motors are protein complexes that transform chemical or electrochemical energy into mechanical work. There are five known rotary molecular motors in nature; the bacterial flagellar motor, and two motors in each of ATP-synthase and V-ATPase. Rotation of the flagellar motor drives a helical propeller that powers bacterial swimming. The function of the other rotary motors is to couple electrochemical ion gradients to synthesis or hydrolysis of ATP, and rotation is a detail of the coupling mechanism rather than the ultimate purpose of the motors. Much has been learned about the mechanism of the F1 part of ATP-synthase and the flagellar motor by measuring the rotation of single motors with a variety of techniques under a wide range of conditions. This chapter will review the structures of ATP-synthase and the flagellar motor, and what has been learned about their mechanisms using single molecule techniques.