Biology subjects

Kinosita, Y.

Publications and source records attributed to Kinosita, Y..

4 recordsLinked to original sources

bioRxiv2020

Distinct chemotactic behavior in the original Escherichia coli K-12 depending on forward-and-backward swimming, not on run-tumble movements

Most motile bacteria are propelled by rigid, helical, flagellar filaments and display distinct swimming patterns to explore their favorable environments. Escherichia coli cells have a reversible rotary motor at the base of each filament. They exhibit a run-tumble swimming pattern, driven by switching of rotatory direction which causes polymorphic flagellar transformation. Here we report a novel swimming mode in E. coli ATCC10798, which is one of the original K-12 clones. High-speed tracking of single ATCC10798 cells showed forward and backward swimming with an average turning angle of 150{degrees}. The flagellar helicity remained right-handed with a 1.3 m pitch and 0.14 m helix radius, which is assumed to be a curly type, regardless of motor switching; the flagella of ATCC10798 did not show polymorphic transformation. The torque and rotational switching of the motor was almost identical to the E. coli W3110 strain, which is a derivative of K-12 and a wild-type for chemotaxis. The single point mutation of N87K in FliC, one of the filament subunits, is critical to the change in flagellar morphology and swimming pattern, and lack of flagellar polymorphism. E. coli cells expressing FliC(N87K) sensed ascending a chemotactic gradient in liquid but did not form rings on a semi-solid surface. Based on these findings, we propose a flagellar polymorphism-dependent migration mechanism in structured environments.

microbiology↗

bioRxiv2020

Swimming ghosts of the halophilic archaeon, Haloferax volcanii

Motility is seen across all domains of life1. Prokaryotes exhibit various types of motilities, such as gliding, swimming, and twitching, driven by supramolecular motility machinery composed of multiple different proteins2. In archaea only swimming motility is reported, driven by the archaellum (archaeal flagellum), a reversible rotary motor consisting of a torque-generating motor and a helical filament which acts as a propeller3,4. Unlike the bacterial flagellar motor (BFM), adenosine triphosphate (ATP) hydrolysis probably drives both motor rotation and filamentous assembly in the archaellum5,6. However, direct evidence is still lacking due to the lack of a versatile model system. Here we present a membrane-permeabilized ghost system that enables the manipulation of intracellular contents, analogous to the triton model in eukaryotic flagella7 and gliding Mycoplasma8,9. We observed high nucleotide selectivity for ATP driving motor rotation, negative cooperativity in ATP hydrolysis and the energetic requirement for at least 12 ATP molecules to be hydrolyzed per revolution of the motor. The response regulator CheY increased motor switching from counterclockwise (CCW) to clockwise (CW) rotation, which is the opposite of a previous report10. Finally, we constructed the torque-speed curve at various [ATP]s and discuss rotary models in which the archaellum has characteristics of both the BFM and F1-ATPase. Because archaea share similar cell division and chemotaxis machinery with other domains of life11,12, our ghost model will be an important tool for the exploration of the universality, diversity, and evolution of biomolecular machinery.

microbiology↗

bioRxiv2018

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. This exceptionally prominent experimental system has allowed us to conclude that the energy source for M. mobile motility is adenosine triphosphate (ATP), and the gliding is largely comprised of repetitions of unitary steps of about 70 nm. In the present study, we show a new motility mode, in which the ghost model prepared with a high concentration of detergent exhibits directed rotational motions with a constant speed. With a rotational speed and viscous friction of a single ghost, the torque was estimated to be [~]30 pN nm at saturated [ATP]s. Although the origin of the rotation has not been conclusively settled, we found that rotary ghosts treated with sialyllactose, the binding target for leg proteins, were stopped. This result suggested that biomolecules embedded on the cell membrane nonspecifically attaches to the glass and works as a flexible pivot point, and the linear motion of the leg is a driving force for a rotary motion. This simple geometry exemplifies the new mechanism, by which the movement of a linear motor is efficiently converted to a constant rotation of the object on a micrometer scale.

biophysics↗

bioRxiv2017

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 {micro}m/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 on sialylated oligosaccharides fixed to solid surfaces. In the present 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 pN following the addition of 0-0.5 mM free sialyllactose (a sialylated oligosaccharide), with a decrease in the number of working units. Following 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 pN. 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.\n\nSIGNIFICANCEMycoplasma is a genus of bacteria that parasitizes animals. Dozens of Mycoplasma species glide over the tissues of their hosts during infection. The gliding machinery of Mycoplasma mobile, the fastest species, includes intracellular motors and hundreds of legs on the cell surface. In the present study, we precisely measured force generation using a highly focused laser beam arrangement (referred to as optical tweezers) under various conditions. The measurements obtained in this study suggest that the rapid gliding exhibited by M. mobile arises from the large gear ratio of its gliding machinery.

biophysics↗