Yoshiaki Kinosita

Direct observation of rotation and steps of the archaellum in the swimming halophilic archaeon Halobacterium salinarum.

Motile archaea swim using a rotary filament, the archaellum, a surface appendage that resembles bacterial flagella structurally, but is homologous to bacterial type IV pili. Little is known about the mechanism by which archaella produce motility. To gain insights into this mechanism, we characterized archaellar function in the model organism Halobacterium salinarum. Three-dimensional tracking of quantum dots enabled visualization of the left-handed corkscrewing of archaea in detail. An advanced analysis method combined with total internal reflection fluorescence microscopy, termed cross-kymography, was developed and revealed a right-handed helical structure of archaella with a rotation speed of 23 ± 5 Hz. Using these structural and kinetic parameters, we computationally reproduced the swimming and precession motion with a hydrodynamic model and estimated the archaellar motor torque to be 50 pN nm. Finally, in a tethered-cell assay, we observed intermittent pauses during rotation with ∼36° or 60° intervals, which we speculate may be a unitary step consuming a single adenosine triphosphate molecule, which supplies chemical energy of 80 pN nm when hydrolysed. From an estimate of the energy input as ten or six adenosine triphosphates per revolution, the efficiency of the motor is calculated to be ∼6–10%.

Three-Dimensional Superlocalization Imaging of Gliding Mycoplasma mobile by Extraordinary Light Transmission through Arrayed Nanoholes.

In this paper, we describe super-resolved sampling of live bacteria based on extraordinary optical transmission (EOT) of light. EOT is produced by surface plasmon confinement and coupling with nanostructures. Bacterial fluorescence is excited by the localized fields for subdiffraction-limited sampling. The concept was applied to elucidating bacterial dynamics of gliding Mycoplasma mobile (M. mobile). The results analyzed with multiple M. mobile bacteria show individual characters and reveal that M. mobile undergoes a significant axial variation at 94 nm. The sampling error of the method is estimated to be much smaller than 1/10 of the diffraction limit both in the lateral and depth axis. The method provides a powerful tool for investigation of biomolecular dynamics at subwavelength precision.

Unitary step of gliding machinery in Mycoplasma mobile

Significance The mechanism of movement of bacteria shows extensive diversity, and some bacteria glide on the substrate surface via an unknown process. Mycoplasma mobile is one of the fastest, exhibiting smooth gliding movement with a speed of 2.0–4.5 µm/s. By applying the modified in vitro ghost model of Mycoplasma mobile to high precision colocalization microscopy, steps of the regular size, ∼70 nm, were detected for the first time in bacteria, to our knowledge. The binding target of the gliding machinery, sialylated oligosaccharides, was expected to be randomly oriented on the surface and, thus, our results suggest that the machinery can drive the steps with a cycle of attachment and detachment even if there is no periodic structure on the substrate. Among the bacteria that glide on substrate surfaces, Mycoplasma mobile is one of the fastest, exhibiting smooth movement with a speed of 2.0–4.5 μm⋅s−1 with a cycle of attachment to and detachment from sialylated oligosaccharides. To study the gliding mechanism at the molecular level, we applied an assay with a fluorescently labeled and membrane-permeabilized ghost model, and investigated the motility by high precision colocalization microscopy. Under conditions designed to reduce the number of motor interactions on a randomly oriented substrate, ghosts took unitary 70-nm steps in the direction of gliding. Although it remains possible that the stepping behavior is produced by multiple interactions, our data suggest that these steps are produced by a unitary gliding machine that need not move between sites arranged on a cytoskeletal lattice.

Detailed Analyses of Stall Force Generation in Mycoplasma mobile Gliding

Mycoplasma mobile is a bacterium that uses a unique mechanism to glide on solid surfaces at a velocity of up to 4.5 μm/s. Its gliding machinery comprises hundreds of units that generate the force for gliding based on the energy derived from ATP; the units catch and pull sialylated oligosaccharides fixed to solid surfaces. In this study, we measured the stall force of wild-type and mutant strains of M. mobile carrying a bead manipulated using optical tweezers. The strains that had been enhanced for binding exhibited weaker stall forces than the wild-type strain, indicating that stall force is related to force generation rather than to binding. The stall force of the wild-type strain decreased linearly from 113 to 19 picoNewtons after the addition of 0–0.5 mM free sialyllactose (a sialylated oligosaccharide), with a decrease in the number of working units. After the addition of 0.5 mM sialyllactose, the cells carrying a bead loaded using optical tweezers exhibited stepwise movements with force increments. The force increments ranged from 1 to 2 picoNewtons. Considering the 70-nm step size, this small-unit force may be explained by the large gear ratio involved in the M. mobile gliding machinery.

Linear motor driven-rotary motion of a membrane-permeabilized ghost in Mycoplasma mobile

Mycoplasma mobile exhibits a smooth gliding movement as does its membrane-permeabilized ghost model. Ghost experiments revealed that the energy source for M. mobile motility is adenosine triphosphate (ATP) and that the gliding comprises repetitions of 70 nm steps. Here we show a new motility mode, in which the ghost model prepared with 0.013% Triton X-100 exhibits directed rotational motions with an average speed of approximately 2.1 Hz when ATP concentration is greater than 3.0 × 10−1 mM. We found that rotary ghosts treated with sialyllactose, the binding target for leg proteins, were stopped. Although the origin of the rotation has not been conclusively determined, this result suggested that biomolecules embedded on the cell membrane nonspecifically attach to the glass and work as a fluid pivot point and that the linear motion of the leg is a driving force for the rotary motion. This simple geometry exemplifies the new motility mode, by which the movement of a linear motor is efficiently converted to a constant rotation of the object on a micrometer scale.

Cross-kymography analysis to simultaneously quantify the function and morphology of the archaellum

In many microorganisms helical structures are important for motility, e.g., bacterial flagella and kink propagation in Spiroplasma eriocheiris. Motile archaea also form a helical-shaped filament called the ‘archaellum’ that is functionally equivalent to the bacterial flagellum, but structurally resembles type IV pili. The archaellum motor consists of 6–8 proteins called fla accessory genes, and the filament assembly is driven by ATP hydrolysis at catalytic sites in FlaI. Remarkably, previous research using a dark-field microscopy showed that right-handed filaments propelled archaeal cells forwards or backwards by clockwise or counterclockwise rotation, respectively. However, the shape and rotational rate of the archaellum during swimming remained unclear, due to the low signal and lack of temporal resolution. Additionally, the structure and the motor properties of the archaellum and bacterial flagellum have not been precisely determined during swimming because they move freely in three-dimensional space. Recently, we developed an advanced method called “cross-kymography analysis”, which enables us to be a long-term observation and simultaneously quantify the function and morphology of helical structures using a total internal reflection fluorescence microscope. In this review, we introduce the basic idea of this analysis, and summarize the latest information in structural and functional characterization of the archaellum motor.

Plasmonic nanohole-based sub-diffraction-limited fluorescence microscopy for imaging of gliding biomolecules

In this presentation, we explore the feasibility of plasmonic nanohole-based sub-diffraction-limited nanoscopy for biomolecular imaging. The technique utilizes near-field distribution localized by surface plasmon localization on metallic nanoholes which is used to sample molecular fluorescence. The optimum geometry of nanohole arrays was determined by numerical analysis. The localization sampling was applied to reconstructing sub-diffraction-limited images of gliding microtubules with a 76 nm effective resolution in the lateral direction. Extraordinary light transmission was also employed to address enhancement of axial resolution using nanohole arrays, based on which extraction of gliding motions of bacteria was demonstrated with an axial resolution down to 50 nm.