RESUMEN
Most motile bacteria use supramolecular motility machinery called bacterial flagellum, which converts the chemical energy gained from ion flux into mechanical rotation. Bacterial cells sense their external environment through a two-component regulatory system consisting of a histidine kinase and response regulator. Combining these systems allows the cells to move toward favorable environments and away from their repellents. A representative example of flagellar motility is run-and-tumble swimming in Escherichia coli, where the counter-clockwise (CCW) rotation of a flagellar bundle propels the cell forward, and the clockwise (CW) rotation undergoes cell re-orientation (tumbling) upon switching the direction of flagellar motor rotation from CCW to CW. In this mini review, we focus on several types of chemotactic behaviors that respond to changes in flagellar shape and direction of rotation. Moreover, our single-cell analysis demonstrated back-and-forth swimming motility of an original E. coli strain. We propose that polymorphic flagellar changes are required to enhance bacterial movement in a structured environment as a colony spread on an agar plate.
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Single-molecular techniques have characterized dynamics of molecular motors such as flagellum in bacteria and myosin, kinesin, and dynein in eukaryotes. We can apply these techniques to a motility machine of archaea, namely, the archaellum, composed of a thin helical filament and a rotary motor. Although the size of the motor hinders the characterization of its motor function under a conventional optical microscope, fluorescence-labeling techniques allow us to visualize the architecture and function of the archaellar filaments in real time. Furthermore, a tiny polystyrene bead attached to the filament enables the visualization of motor rotation through the bead rotation and quantification of biophysical properties such as speed and torque produced by the rotary motor imbedded in the cell membrane. In this chapter, I describe the details of the above biophysical method based on an optical microscope.
Asunto(s)
Microscopía , Proteínas Motoras Moleculares , Proteínas Motoras Moleculares/metabolismo , Rotación , Dineínas/metabolismo , Bacterias/metabolismo , Flagelos/metabolismoRESUMEN
Mycoplasma mobile is one of the fastest gliding bacteria, gliding with a speed of 4.5 µm s-1. This gliding motility is driven by a concerted movement of 450 supramolecular motor units composed of three proteins, Gli123, Gli349, and Gli521, in the gliding motility machinery. With general experimental setups, it is difficult to obtain the information on how each motor unit works. This chapter describes strategies to decrease the number of active motor units to extract stepwise cell movements driven by a minimum number of motor units. We also describe an unforeseen motility mode in which the leg motions convert the gliding motion into rotary motion, which enables us to characterize the motor torque and energy-conversion efficiency by adding some more assumptions.
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Proteínas Bacterianas , Mycoplasma , Proteínas Bacterianas/metabolismo , Rotación , Mycoplasma/metabolismo , MovimientoRESUMEN
Archaea swim using the archaellum (archaeal flagellum), a reversible rotary motor consisting of a torque-generating motor and a helical filament, which acts as a propeller. Unlike the bacterial flagellar motor (BFM), ATP (adenosine-5'-triphosphate) hydrolysis probably drives both motor rotation and filamentous assembly in the archaellum. 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 flagella and gliding Mycoplasma 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. 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 life, our ghost model will be an important tool for the exploration of the universality, diversity, and evolution of biomolecular machinery.
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Membrana Celular , Quimiotaxis/fisiología , Haloferax volcanii , Modelos Biológicos , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular , Flagelos/química , Flagelos/metabolismo , Haloferax volcanii/citología , Haloferax volcanii/metabolismo , Cinética , Proteínas Quimiotácticas Aceptoras de Metilo/química , Proteínas Quimiotácticas Aceptoras de Metilo/metabolismo , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/metabolismoRESUMEN
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 the rotational 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°. The flagellar helicity remained right-handed with a 1.3 µm pitch and 0.14 µm helix radius, which is consistent with the feature of 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 spread on a semi-solid surface. Based on these results, we concluded that a flagellar polymorphism is essential for spreading in structured environments.
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Quimiotaxis/fisiología , Escherichia coli K12/fisiología , Flagelos/fisiología , Modelos Biológicos , MutaciónRESUMEN
Motility often plays a decisive role in the survival of species. Five systems of motility have been studied in depth: those propelled by bacterial flagella, eukaryotic actin polymerization and the eukaryotic motor proteins myosin, kinesin and dynein. However, many organisms exhibit surprisingly diverse motilities, and advances in genomics, molecular biology and imaging have showed that those motilities have inherently independent mechanisms. This makes defining the breadth of motility nontrivial, because novel motilities may be driven by unknown mechanisms. Here, we classify the known motilities based on the unique classes of movement-producing protein architectures. Based on this criterion, the current total of independent motility systems stands at 18 types. In this perspective, we discuss these modes of motility relative to the latest phylogenetic Tree of Life and propose a history of motility. During the ~4 billion years since the emergence of life, motility arose in Bacteria with flagella and pili, and in Archaea with archaella. Newer modes of motility became possible in Eukarya with changes to the cell envelope. Presence or absence of a peptidoglycan layer, the acquisition of robust membrane dynamics, the enlargement of cells and environmental opportunities likely provided the context for the (co)evolution of novel types of motility.
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Movimiento Celular/genética , Movimiento Celular/fisiología , Flagelos/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Animales , Bacterias , Evolución Biológica , Dineínas/metabolismo , Evolución Molecular , Flagelos/genética , Humanos , Cinesinas/metabolismo , Miosinas/metabolismo , FilogeniaRESUMEN
It is unknown how the archaellum-the rotary propeller used by Archaea for motility-works. To further understand the molecular mechanism by which the hexameric ATPase motor protein FlaI drives rotation of the membrane-embedded archaellar motor, we determined motor torque by imposition of various loads on Halobacterium salinarum archaella. Markers of different sizes were attached to single archaella, and their trajectories were quantified using three-dimensional tracking and high-speed recording. We show that rotation slows as the viscous drag of markers increases, but torque remains constant at 160 pN·nm independent of rotation speed. Notably, the estimated work done in a single rotation is twice the expected energy that would come from hydrolysis of six ATP molecules in the hexamer, indicating that more ATP molecules are required for one rotation of archaellum. To reconcile the apparent contradiction, we suggest a new and general model for the mechanism of ATP-driven rotary motors.
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Proteínas Arqueales/fisiología , Flagelos/fisiología , Halobacterium salinarum/fisiología , Proteínas Motoras Moleculares/fisiología , ATPasas de Translocación de Protón/fisiología , Adenosina Trifosfato/metabolismo , Dominio Catalítico , Hidrólisis , Microscopía , Modelos Moleculares , Rotación , Torque , ViscosidadRESUMEN
Bacteria and archaea exhibit tactical behavior and can move up and down chemical gradients. This tactical behavior relies on a motility structure, which is guided by a chemosensory system. Environmental signals are sensed by membrane-inserted chemosensory receptors that are organized in large ordered arrays. While the cellular positioning of the chemotaxis machinery and that of the flagellum have been studied in detail in bacteria, we have little knowledge about the localization of such macromolecular assemblies in archaea. Although the archaeal motility structure, the archaellum, is fundamentally different from the flagellum, archaea have received the chemosensory machinery from bacteria and have connected this system with the archaellum. Here, we applied a combination of time-lapse imaging and fluorescence and electron microscopy using the model euryarchaeon Haloferax volcanii and found that archaella were specifically present at the cell poles of actively dividing rod-shaped cells. The chemosensory arrays also had a polar preference, but in addition, several smaller arrays moved freely in the lateral membranes. In the stationary phase, rod-shaped cells became round and chemosensory arrays were disassembled. The positioning of archaella and that of chemosensory arrays are not interdependent and likely require an independent form of positioning machinery. This work showed that, in the rod-shaped haloarchaeal cells, the positioning of the archaellum and of the chemosensory arrays is regulated in time and in space. These insights into the cellular organization of H. volcanii suggest the presence of an active mechanism responsible for the positioning of macromolecular protein complexes in archaea.IMPORTANCE Archaea are ubiquitous single cellular microorganisms that play important ecological roles in nature. The intracellular organization of archaeal cells is among the unresolved mysteries of archaeal biology. With this work, we show that cells of haloarchaea are polarized. The cellular positioning of proteins involved in chemotaxis and motility is spatially and temporally organized in these cells. This suggests the presence of a specific mechanism responsible for the positioning of macromolecular protein complexes in archaea.
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Proteínas Arqueales/química , Polaridad Celular , Quimiotaxis , Haloferax volcanii/fisiología , Citoplasma/química , Flagelos/fisiología , Haloferax volcanii/ultraestructura , Microscopía Electrónica , Imagen de Lapso de TiempoRESUMEN
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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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.
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Movimiento/fisiología , Mycoplasma/metabolismo , Mycoplasma/fisiología , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/metabolismo , Membrana Eritrocítica/metabolismo , Membranas/metabolismo , Proteínas Motoras Moleculares/metabolismo , RotaciónRESUMEN
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.
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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.
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Fenómenos Mecánicos , Mycoplasma , Fenómenos Biomecánicos , Estrés Mecánico , Propiedades de SuperficieRESUMEN
A bean bug symbiont, Burkholderia sp. RPE64, selectively colonizes the gut crypts by flagella-mediated motility: however, the mechanism for this colonization remains unclear. Here, to obtain clues to this mechanism, we characterized the swimming motility of the Burkholderia symbiont under an advanced optical microscope. High-speed imaging of cells enabled the detection of turn events with up to 5-ms temporal resolution, indicating that cells showed reversal motions (θ ~ 180°) with rapid changes in speed by a factor of 3.6. Remarkably, staining of the flagellar filaments with a fluorescent dye Cy3 revealed that the flagellar filaments wrap around the cell body with a motion like that of a ribbon streamer in rhythmic gymnastics. A motility assay with total internal reflection fluorescence microscopy revealed that the left-handed flagellum wound around the cell body and propelled it forward by its clockwise rotation. We also detected periodic-fluorescent signals of flagella on the glass surface, suggesting that flagella possibly contacted the solid surface directly and produced a gliding-like motion driven by flagellar rotation. Finally, the wrapping motion was also observed in a symbiotic bacterium of the bobtail squid, Aliivibrio fischeri, suggesting that this motility mode may contribute to migration on the mucus-filled narrow passage connecting to the symbiotic organ.
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Burkholderia/fisiología , Movimiento Celular/fisiología , Flagelos/fisiología , Animales , Cuerpo Celular/fisiología , Ensayos de Migración Celular , Células CultivadasRESUMEN
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 ± 5â 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 50â pNâ nm. Finally, in a tethered-cell assay, we observed intermittent pauses during rotation with â¼36° or 60° intervals, which we speculate may be a unitary step consuming a single adenosine triphosphate molecule, which supplies chemical energy of 80â pNâ 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 â¼6-10%.
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Fimbrias Bacterianas/fisiología , Halobacterium salinarum/citología , Halobacterium salinarum/fisiología , Fimbrias Bacterianas/química , Flagelos/química , Flagelos/fisiología , Halobacterium salinarum/química , Halobacterium salinarum/ultraestructura , Cinética , Microscopía Fluorescente/métodos , Proteínas Motoras Moleculares , Movimiento , Puntos Cuánticos , Rotación , TorqueRESUMEN
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.
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Imagenología Tridimensional/métodos , Luz , Mycoplasma/fisiología , Nanoestructuras/química , Nanoestructuras/ultraestructuraRESUMEN
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 µmâ 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.