Ion selectivity and rotor coupling of the Vibrio flagellar sodium-driven stator unit.

Bacteria swim using a flagellar motor that is powered by stator units. Vibrio spp. are highly motile bacteria responsible for various human diseases, the polar flagella of which are exclusively driven by sodium-dependent stator units (PomAB). However, how ion selectivity is attained, how ion transport triggers the directional rotation of the stator unit, and how the stator unit is incorporated into the flagellar rotor remained largely unclear. Here, we have determined by cryo-electron microscopy the structure of Vibrio PomAB. The electrostatic potential map uncovers sodium binding sites, which together with functional experiments and molecular dynamics simulations, reveal a mechanism for ion translocation and selectivity. Bulky hydrophobic residues from PomA prime PomA for clockwise rotation. We propose that a dynamic helical motif in PomA regulates the distance between PomA subunit cytoplasmic domains, stator unit activation, and torque transmission. Together, our study provides mechanistic insights for understanding ion selectivity and rotor incorporation of the stator unit of the bacterial flagellum.

A multi-state dynamic process confers mechano-adaptation to a biological nanomachine.

Adaptation is a defining feature of living systems. The bacterial flagellar motor adapts to changes in the external mechanical load by adding or removing torque-generating (stator) units. But the molecular mechanism behind this mechano-adaptation remains unclear. Here, we combine single motor eletrorotation experiments and theoretical modeling to show that mechano-adaptation of the flagellar motor is enabled by multiple mechanosensitive internal states. Dwell time statistics from experiments suggest the existence of at least two bound states with a high and a low unbinding rate, respectively. A first-passage-time analysis of a four-state model quantitatively explains the experimental data and determines the transition rates among all four states. The torque generated by bound stator units controls their effective unbinding rate by modulating the transition between the bound states, possibly via a catch bond mechanism. Similar force-mediated feedback enabled by multiple internal states may apply to adaptation in other macromolecular complexes.

Structural basis of torque generation in the bi-directional bacterial flagellar motor.

The flagellar stator unit is an oligomeric complex of two membrane proteins (MotA5B2) that powers bi-directional rotation of the bacterial flagellum. Harnessing the ion motive force across the cytoplasmic membrane, the stator unit operates as a miniature rotary motor itself to provide torque for rotation of the flagellum. Recent cryo-electron microscopic (cryo-EM) structures of the stator unit provided novel insights into its assembly, function, and subunit stoichiometry, revealing the ion flux pathway and the torque generation mechanism. Furthermore, in situ cryo-electron tomography (cryo-ET) studies revealed unprecedented details of the interactions between stator unit and rotor. In this review, we summarize recent advances in our understanding of the structure and function of the flagellar stator unit, torque generation, and directional switching of the motor.

Bacterial motility: machinery and mechanisms.

Bacteria have developed a large array of motility mechanisms to exploit available resources and environments. These mechanisms can be broadly classified into swimming in aqueous media and movement over solid surfaces. Swimming motility involves either the rotation of rigid helical filaments through the external medium or gyration of the cell body in response to the rotation of internal filaments. On surfaces, bacteria swarm collectively in a thin layer of fluid powered by the rotation of rigid helical filaments, they twitch by assembling and disassembling type IV pili, they glide by driving adhesins along tracks fixed to the cell surface and, finally, non-motile cells slide over surfaces in response to outward forces due to colony growth. Recent technological advances, especially in cryo-electron microscopy, have greatly improved our knowledge of the molecular machinery that powers the various forms of bacterial motility. In this Review, we describe the current understanding of the physical and molecular mechanisms that allow bacteria to move around.

Mechanosensitive remodeling of the bacterial flagellar motor is independent of direction of rotation.

Motility is important for the survival and dispersal of many bacteria, and it often plays a role during infections. Regulation of bacterial motility by chemical stimuli is well studied, but recent work has added a new dimension to the problem of motility control. The bidirectional flagellar motor of the bacterium Escherichia coli recruits or releases torque-generating units (stator units) in response to changes in load. Here, we show that this mechanosensitive remodeling of the flagellar motor is independent of direction of rotation. Remodeling rate constants in clockwise rotating motors and in counterclockwise rotating motors, measured previously, fall on the same curve if plotted against torque. Increased torque decreases the off rate of stator units from the motor, thereby increasing the number of active stator units at steady state. A simple mathematical model based on observed dynamics provides quantitative insight into the underlying molecular interactions. The torque-dependent remodeling mechanism represents a robust strategy to quickly regulate output (torque) in response to changes in demand (load).

Structure and Function of Stator Units of the Bacterial Flagellar Motor.

Many bacteria use the flagellum for locomotion and chemotaxis. Its bidirectional rotation is driven by a membrane-embedded motor, which uses energy from the transmembrane ion gradient to generate torque at the interface between stator units and rotor. The structural organization of the stator unit (MotAB), its conformational changes upon ion transport, and how these changes power rotation of the flagellum remain unknown. Here, we present ~3 Å-resolution cryoelectron microscopy reconstructions of the stator unit in different functional states. We show that the stator unit consists of a dimer of MotB surrounded by a pentamer of MotA. Combining structural data with mutagenesis and functional studies, we identify key residues involved in torque generation and present a detailed mechanistic model for motor function and switching of rotational direction.

Torque-dependent remodeling of the bacterial flagellar motor.

Multisubunit protein complexes are ubiquitous in biology and perform a plethora of essential functions. Most of the scientific literature treats such assemblies as static: their function is assumed to be independent of their manner of assembly, and their structure is assumed to remain intact until they are degraded. Recent observations of the bacterial flagellar motor, among others, bring these notions into question. The torque-generating stator units of the motor assemble and disassemble in response to changes in load. Here, we used electrorotation to drive tethered cells forward, which decreases motor load, and measured the resulting stator dynamics. No disassembly occurred while the torque remained high, but all of the stator units were released when the motor was spun near the zero-torque speed. When the electrorotation was turned off, so that the load was again high, stator units were recruited, increasing motor speed in a stepwise fashion. A model in which speed affects the binding rate and torque affects the free energy of bound stator units captures the observed torque-dependent stator assembly dynamics, providing a quantitative framework for the environmentally regulated self-assembly of a major macromolecular machine.

Size structures sensory hierarchy in ocean life.

Survival in aquatic environments requires organisms to have effective means of collecting information from their surroundings through various sensing strategies. In this study, we explore how sensing mode and range depend on body size. We find a hierarchy of sensing modes determined by body size. With increasing body size, a larger battery of modes becomes available (chemosensing, mechanosensing, vision, hearing and echolocation, in that order) while the sensing range also increases. This size-dependent hierarchy and the transitions between primary sensory modes are explained on the grounds of limiting factors set by physiology and the physical laws governing signal generation, transmission and reception. We theoretically predict the body size limits for various sensory modes, which align well with size ranges found in literature. The treatise of all ocean life, from unicellular organisms to whales, demonstrates how body size determines available sensing modes, and thereby acts as a major structuring factor of aquatic life.

Quiet swimming at low Reynolds number.

The stresslet provides a simple model of the flow created by a small, freely swimming and neutrally buoyant aquatic organism and shows that the far field fluid disturbance created by such an organism in general decays as one over distance squared. Here we discuss a quieter swimming mode that eliminates the stresslet component of the flow and leads to a faster spatial decay of the fluid disturbance described by a force quadrupole that decays as one over distance cubed. Motivated by recent experimental results on fluid disturbances due to small aquatic organisms, we demonstrate that a three-Stokeslet model of a swimming organism which uses breast stroke type kinematics is an example of such a quiet swimmer. We show that the fluid disturbance in both the near field and the far field is significantly reduced by appropriately arranging the propulsion apparatus, and we find that the far field power laws are valid surprisingly close to the organism. Finally, we discuss point force models as a general framework for hypothesis generation and experimental exploration of fluid mediated predator-prey interactions in the planktonic world.

Flow disturbances generated by feeding and swimming zooplankton.

Interactions between planktonic organisms, such as detection of prey, predators, and mates, are often mediated by fluid signals. Consequently, many plankton predators perceive their prey from the fluid disturbances that it generates when it feeds and swims. Zooplankton should therefore seek to minimize the fluid disturbance that they produce. By means of particle image velocimetry, we describe the fluid disturbances produced by feeding and swimming in zooplankton with diverse propulsion mechanisms and ranging from 10-µm flagellates to greater than millimeter-sized copepods. We show that zooplankton, in which feeding and swimming are separate processes, produce flow disturbances during swimming with a much faster spatial attenuation (velocity u varies with distance r as u ∝ r(-3) to r(-4)) than that produced by zooplankton for which feeding and propulsion are the same process (u ∝ r(-1) to r(-2)). As a result, the spatial extension of the fluid disturbance produced by swimmers is an order of magnitude smaller than that produced by feeders at similar Reynolds numbers. The "quiet" propulsion of swimmers is achieved either through swimming erratically by short-lasting power strokes, generating viscous vortex rings, or by "breast-stroke swimming." Both produce rapidly attenuating flows. The more "noisy" swimming of those that are constrained by a need to simultaneously feed is due to constantly beating flagella or appendages that are positioned either anteriorly or posteriorly on the (cell) body. These patterns transcend differences in size and taxonomy and have thus evolved multiple times, suggesting a strong selective pressure to minimize predation risk.

Hydrodynamics and energetics of jumping copepod nauplii and copepodids.

Within its life cycle, a copepod goes through drastic changes in size, shape and swimming mode. In particular, there is a stark difference between the early (nauplius) and later (copepodid) stages. Copepods inhabit an intermediate Reynolds number regime (between ~1 and 100) where both viscosity and inertia are potentially important, and the Reynolds number changes by an order of magnitude during growth. Thus we expect the life stage related changes experienced by a copepod to result in hydrodynamic and energetic differences, ultimately affecting the fitness. To quantify these differences, we measured the swimming kinematics and fluid flow around jumping Acartia tonsa at different stages of its life cycle, using particle image velocimetry and particle tracking velocimetry. We found that the flow structures around nauplii and copepodids are topologically different, with one and two vortex rings, respectively. Our measurements suggest that copepodids cover a larger distance compared to their body size in each jump and are also hydrodynamically quieter, as the flow disturbance they create attenuates faster with distance. Also, copepodids are energetically more efficient than nauplii, presumably due to the change in hydrodynamic regime accompanied with a well-adapted body form and swimming stroke.

Noncoalescence in the oblique collision of fluid jets.

When two jets of fluid collide, they can "bounce" off each other, due to a thin film of air which keeps them separated. We describe the phenomenon of stable noncoalescence between two jets of the same fluid, colliding obliquely with each other. Using a simple experimental setup, we carry out a parametric study of the bouncing jets by varying the jet diameter, velocity, angle of inclination, and fluid viscosity, which suggests that the contact time of bouncing jets scales as the square root of the normal Weber number We. A dimensionless parameter K = (We sqrt[Re]/sinα)(1/2), where Re is the normal Reynolds number and α the angle of inclination of the jets, quantitatively captures the transition of colliding jets from bouncing to coalescence. This parameter draws parallels between jet coalescence and droplet splashing and indicates that the transition is governed by a surface instability. Stable and continuous noncoalescence between fluid jets makes it a good platform for experimental studies of the interaction between fluid interfaces and the properties of the interfacial air films.