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1.
Opt Express ; 31(4): 5167-5180, 2023 Feb 13.
Artículo en Inglés | MEDLINE | ID: mdl-36823805

RESUMEN

We propose a simple, cost-effective method for synchronized phase contrast and fluorescence video acquisition in live samples. Counter-phased pulses of phase contrast illumination and fluorescence excitation light are synchronized with the exposure of the two fields of an interlaced camera sensor. This results in a video sequence in which each frame contains both exposure modes, each in half of its pixels. The method allows real-time acquisition and display of synchronized and spatially aligned phase contrast and fluorescence image sequences that can be separated by de-interlacing in two independent videos. The method can be implemented on any fluorescence microscope with a camera port without needing to modify the optical path.

2.
Nat Commun ; 13(1): 5327, 2022 09 10.
Artículo en Inglés | MEDLINE | ID: mdl-36088344

RESUMEN

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.


Asunto(s)
Flagelos , Proteínas Motoras Moleculares , Aclimatación , Bacterias/metabolismo , Flagelos/metabolismo , Proteínas Motoras Moleculares/metabolismo , Torque
3.
Proc Natl Acad Sci U S A ; 119(6)2022 02 08.
Artículo en Inglés | MEDLINE | ID: mdl-35131853

RESUMEN

Bacterial cells interact with solid surfaces and change their lifestyle from single free-swimming cells to sessile communal structures (biofilms). Cyclic di-guanosine monophosphate (c-di-GMP) is central to this process, yet we lack tools for direct dynamic visualization of c-di-GMP in single cells. Here, we developed a fluorescent protein-based c-di-GMP-sensing system for Escherichia coli that allowed us to visualize initial signaling events and assess the role played by the flagellar motor. The sensor was pH sensitive, and the events that appeared on a seconds' timescale were alkaline spikes in the intracellular pH. These spikes were not apparent when signals from different cells were averaged. Instead, a signal appeared on a minutes' timescale that proved to be due to an increase in intracellular c-di-GMP. This increase, but not the alkaline spikes, depended upon a functional flagellar motor. The kinetics and the amplitude of both the pH and c-di-GMP responses displayed cell-to-cell variability indicative of the distinct ways the cells approached and interacted with the surface. The energetic status of a cell can modulate these events. In particular, the alkaline spikes displayed an oscillatory behavior and the c-di-GMP increase was modest in the presence of glucose.


Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Transducción de Señal/fisiología , GMP Cíclico/metabolismo , Flagelos/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Vidrio , Concentración de Iones de Hidrógeno , Sistemas de Mensajero Secundario/fisiología , Propiedades de Superficie
4.
Trends Biochem Sci ; 47(2): 160-172, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-34294545

RESUMEN

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.


Asunto(s)
Proteínas Bacterianas , Flagelos , Bacterias/metabolismo , Proteínas Bacterianas/química , Microscopía por Crioelectrón/métodos , Flagelos/química , Flagelos/metabolismo , Flagelos/ultraestructura , Torque
5.
Nat Rev Microbiol ; 20(3): 161-173, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-34548639

RESUMEN

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.


Asunto(s)
Fenómenos Fisiológicos Bacterianos , Movimiento/fisiología , Adhesinas Bacterianas/fisiología , Animales , Bacterias , Microscopía por Crioelectrón/métodos , Fimbrias Bacterianas/fisiología
6.
Proc Natl Acad Sci U S A ; 118(15)2021 04 13.
Artículo en Inglés | MEDLINE | ID: mdl-33876769

RESUMEN

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).


Asunto(s)
Flagelos/química , Mecanotransducción Celular , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Escherichia coli , Flagelos/metabolismo , Modelos Teóricos , Rotación
7.
Cell ; 183(1): 244-257.e16, 2020 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-32931735

RESUMEN

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.


Asunto(s)
Proteínas Bacterianas/ultraestructura , Flagelos/ultraestructura , Bacterias/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Microscopía por Crioelectrón/métodos , Flagelos/metabolismo , Conformación Proteica , Torque
8.
Sci Adv ; 6(10): eaay6616, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-32181348

RESUMEN

The gliding bacterium Flavobacterium johnsoniae is known to have an adhesin, SprB, that moves along the cell surface on a spiral track. Following viscous shear, cells can be tethered by the addition of an anti-SprB antibody, causing spinning at 3 Hz. Labeling the type 9 secretion system (T9SS) with a YFP fusion of GldL showed a yellow fluorescent spot near the rotation axis, indicating that the motor driving the motion is associated with the T9SS. The distance between the rotation axis and the track (90 nm) was determined after adding a Cy3 label for SprB. A rotary motor spinning a pinion of radius 90 nm at 3 Hz would cause a spot on its periphery to move at 1.5 µm/s, the gliding speed. We suggest the pinion drives a flexible tread that carries SprB along a track fixed to the cell surface. Cells glide when this adhesin adheres to the solid substratum.


Asunto(s)
Adhesinas Bacterianas/genética , Sistemas de Secreción Bacterianos/genética , Flavobacterium/genética , Adhesinas Bacterianas/metabolismo , Anticuerpos Antibacterianos/química , Adhesión Bacteriana/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos/metabolismo , Fenómenos Biomecánicos , Carbocianinas/química , Flavobacterium/metabolismo , Colorantes Fluorescentes/química , Expresión Génica , Genes Reporteros , Locomoción/fisiología , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Rotación
9.
Proc Natl Acad Sci U S A ; 116(24): 11764-11769, 2019 06 11.
Artículo en Inglés | MEDLINE | ID: mdl-31142644

RESUMEN

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.


Asunto(s)
Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Flagelos/metabolismo , Sustancias Macromoleculares/metabolismo , Proteínas Motoras Moleculares/metabolismo , Torque
10.
Proc Natl Acad Sci U S A ; 115(34): 8633-8638, 2018 08 21.
Artículo en Inglés | MEDLINE | ID: mdl-30082394

RESUMEN

The human microbiome is an assemblage of diverse bacteria that interact with one another to form communities. Bacteria in a given community are arranged in a 3D matrix with many degrees of freedom. Snapshots of the community display well-defined structures, but the steps required for their assembly are not understood. Here, we show that this construction is carried out with the help of gliding bacteria. Gliding is defined as the motion of cells over a solid or semisolid surface without the necessity of growth or the aid of pili or flagella. Genomic analysis suggests that gliding bacteria are present in human microbial communities. We focus on Capnocytophaga gingivalis, which is present in abundance in the human oral microbiome. Tracking of fluorescently labeled single cells and of gas bubbles carried by fluid flow shows that swarms of C. gingivalis are layered, with cells in the upper layers moving more rapidly than those in the lower layers. Thus, cells also glide on top of one another. Cells of nonmotile bacterial species attach to the surface of C. gingivalis and are propelled as cargo. The cargo cell moves along the length of a C. gingivalis cell, looping from one pole to the other. Multicolor fluorescent spectral imaging of cells of different live but nonmotile bacterial species reveals their long-range transport in a polymicrobial community. A swarm of C. gingivalis transports some nonmotile bacterial species more efficiently than others and helps to shape the spatial organization of a polymicrobial community.


Asunto(s)
Capnocytophaga/fisiología , Consorcios Microbianos/fisiología , Microbiota/fisiología , Boca/microbiología , Humanos
11.
Methods Mol Biol ; 1729: 71-76, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29429083

RESUMEN

We describe labeling of bacteria with amino-specific or sulfhydryl-specific Alexa Fluor dyes, methods that allow visualization of flagellar filaments, even in swimming cells. Bacterial flagellar filaments are long (~10 µm), but of small diameter (~20 nm), and their rotation rates are high (>100 Hz), so visualization is difficult. Dark-field microscopy works well with isolated filaments, but visualization in situ is hampered by light scattered from cell bodies, which obscures short filaments or the proximal ends of long filaments. Differential interference contrast microscopy also works, but is technically difficult and suffers from a narrow depth of field and low image contrast; background subtraction and contrast enhancement are necessary. If filaments are fluorescent, they can be imaged in their entirety using standard fluorescence microscopes. For imaging in vivo, blurring can be prevented by strobing the light source or by using a camera with a fast shutter. The former method is preferred, since it minimizes bleaching.


Asunto(s)
Bacterias/citología , Flagelos/ultraestructura , Colorantes Fluorescentes/metabolismo , Bacterias/metabolismo , Flagelos/metabolismo , Procesamiento de Imagen Asistido por Computador , Microscopía Fluorescente/métodos , Grabación en Video
12.
Biophys J ; 114(3): 641-649, 2018 02 06.
Artículo en Inglés | MEDLINE | ID: mdl-29414710

RESUMEN

The molecular cascade that controls switching of the direction of rotation of Escherichia coli flagellar motors is well known, but the conformational changes that allow the rotor to switch are still unclear. The signaling molecule CheY, when phosphorylated, binds to the C-ring at the base of the rotor, raising the probability that the motor spins clockwise. When the concentration of CheY-P is so low that the motor rotates exclusively counterclockwise (CCW), the C-ring recruits more monomers of FliM and tetramers of FliN, the proteins to which CheY-P binds, thus increasing the motor's sensitivity to CheY-P and allowing it to switch once again. Motors that rotate exclusively CCW have more FliM and FliN subunits in their C-rings than motors that rotate exclusively clockwise. How are the new subunits accommodated? Does the diameter of the C-ring increase, or do FliM and FliN get packed in a different pattern, keeping the overall diameter of the C-ring constant? Here, by measuring fluorescence anisotropy of yellow fluorescent protein-labeled motors, we show that the CCW C-rings accommodate more FliM monomers without changing the spacing between them, and more FliN monomers at the same time as increasing their effective spacing and/or changing their orientation within the tetrameric structure.


Asunto(s)
Proteínas Bacterianas/química , Escherichia coli/metabolismo , Polarización de Fluorescencia/métodos , Proteínas Quimiotácticas Aceptoras de Metilo/química , Conformación Proteica , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Proteínas Quimiotácticas Aceptoras de Metilo/metabolismo , Fosforilación , Unión Proteica
13.
Science ; 358(6362): 446-447, 2017 10 27.
Artículo en Inglés | MEDLINE | ID: mdl-29074753
14.
Phys Rev E ; 95(6-1): 063106, 2017 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-28709256

RESUMEN

The helical flagella that are attached to the cell body of bacteria such as Escherichia coli and Salmonella typhimurium allow the cell to swim in a fluid environment. These flagella are capable of polymorphic transformation in that they take on various helical shapes that differ in helical pitch, radius, and chirality. We present a mathematical model of a single flagellum described by Kirchhoff rod theory that is immersed in a fluid governed by Stokes equations. We perform numerical simulations to demonstrate two mechanisms by which polymorphic transformation can occur, as observed in experiments. First, we consider a flagellar filament attached to a rotary motor in which transformations are triggered by a reversal of the direction of motor rotation [L. Turner et al., J. Bacteriol. 182, 2793 (2000)10.1128/JB.182.10.2793-2801.2000]. We then consider a filament that is fixed on one end and immersed in an external fluid flow [H. Hotani, J. Mol. Biol. 156, 791 (1982)10.1016/0022-2836(82)90142-5]. The detailed dynamics of the helical flagellum interacting with a viscous fluid is discussed and comparisons with experimental and theoretical results are provided.


Asunto(s)
Bacterias , Flagelos , Modelos Biológicos , Fenómenos Fisiológicos Bacterianos , Simulación por Computador , Movimiento , Rotación , Torsión Mecánica , Sustancias Viscoelásticas , Viscosidad
15.
Protein Sci ; 26(7): 1249-1251, 2017 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-27679984

RESUMEN

A short review is given of recent work showing that the flagellar rotary motor of the bacterium Escherichia coli remodels to match its operating point (the fraction of time that it spins clockwise) to the requirements of the chemotaxis signaling network, and to provide the torque necessary to operate at different viscous loads.


Asunto(s)
Quimiotaxis/fisiología , Escherichia coli/fisiología , Flagelos/fisiología , Transducción de Señal/fisiología
16.
Biophys J ; 111(5): 1008-13, 2016 Sep 06.
Artículo en Inglés | MEDLINE | ID: mdl-27602728

RESUMEN

Flavobacterium johnsoniae, a rod-shaped bacterium, glides over surfaces at speeds of ∼2 µm/s. The propulsion of a cell-surface adhesin, SprB, is known to enable gliding. We used cephalexin to generate elongated cells with irregular shapes and followed their displacement in three dimensions. These cells rolled about their long axes as they moved forward, following a right-handed trajectory. We coated gold nanoparticles with an SprB antibody and tracked them in three dimensions in an evanescent field where the nanoparticles appeared brighter when they were closer to the glass. The nanoparticles followed a right-handed spiral trajectory on the surface of the cell. Thus, if SprB were to adhere to the glass rather than to a nanoparticle, the cell would move forward along a right-handed trajectory, as observed, but in a direction opposite to that of the nanoparticle.


Asunto(s)
Adhesinas Bacterianas/metabolismo , Flavobacterium/fisiología , Movimiento/fisiología , Antibacterianos , Anticuerpos Antibacterianos , Cefalexina , Vidrio , Oro , Nanopartículas del Metal , Microscopía , Movimiento (Física)
17.
Biophys J ; 111(3): 630-639, 2016 Aug 09.
Artículo en Inglés | MEDLINE | ID: mdl-27508446

RESUMEN

A complete description of the swimming behavior of a bacterium requires measurement of the displacement and orientation of the cell body together with a description of the movement of the flagella. We rebuilt a tracking microscope so that we could visualize flagellar filaments of tracked cells by fluorescence. We studied Escherichia coli (cells of various lengths, including swarm cells), Bacillus subtilis (wild-type and a mutant with fewer flagella), and a motile Streptococcus (now Enterococcus). The run-and-tumble statistics were nearly the same regardless of cell shape, length, and flagellation; however, swarm cells rarely tumbled, and cells of Enterococcus tended to swim in loops when moving slowly. There were events in which filaments underwent polymorphic transformations but remained in bundles, leading to small deflections in direction of travel. Tumble speeds were ∼2/3 as large as run speeds, and the rates of change of swimming direction while running or tumbling were smaller when cells swam more rapidly. If a smaller fraction of filaments were involved in tumbles, the tumble intervals were shorter and the angles between runs were smaller.


Asunto(s)
Bacterias/citología , Rastreo Celular , Flagelos/metabolismo
18.
Nat Phys ; 12(2): 175-178, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-27499800

RESUMEN

Caulobacter crescentus, a monotrichous bacterium, swims by rotating a single right-handed helical filament. CW motor rotation thrusts the cell forward 1, a mode of motility known as the pusher mode; CCW motor rotation pulls the cell backward, a mode of motility referred to as the puller mode 2. The situation is opposite in E. coli, a peritrichous bacterium, where CCW rotation of multiple left-handed filaments drives the cell forward. The flagellar motor in E. coli generates more torque in the CCW direction than the CW direction in swimming cells 3,4. However, monotrichous bacteria including C. crescentus swim forward and backward at similar speeds, prompting the assumption that motor torques in the two modes are the same 5,6. Here, we present evidence that motors in C. crescentus develop higher torques in the puller mode than in the pusher mode, and suggest that the anisotropy in torque-generation is similar in two species, despite the differences in filament handedness and motor bias (probability of CW rotation).

19.
Proc Natl Acad Sci U S A ; 113(17): 4783-7, 2016 Apr 26.
Artículo en Inglés | MEDLINE | ID: mdl-27071081

RESUMEN

Most bacteria that swim, including Escherichia coli, are propelled by helical filaments, each driven at its base by a rotary motor powered by a proton or a sodium ion electrochemical gradient. Each motor contains a number of stator complexes, comprising 4MotA 2MotB or 4PomA 2PomB, proteins anchored to the rigid peptidoglycan layer of the cell wall. These proteins exert torque on a rotor that spans the inner membrane. A shaft connected to the rotor passes through the peptidoglycan and the outer membrane through bushings, the P and L rings, connecting to the filament by a flexible coupling known as the hook. Although the external components, the hook and the filament, are known to rotate, having been tethered to glass or marked by latex beads, the rotation of the internal components has remained only a reasonable assumption. Here, by using polarized light to bleach and probe an internal YFP-FliN fusion, we show that the innermost components of the cytoplasmic ring rotate at a rate similar to that of the hook.


Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/ultraestructura , Microscopía Fluorescente/métodos , Imagen Molecular/métodos , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/ultraestructura , Técnicas de Sonda Molecular , Fotoblanqueo , Rotación
20.
Sci Adv ; 1(9): e1500299, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26601280

RESUMEN

Stimulation of Escherichia coli by exponential ramps of chemoattractants generates step changes in the concentration of the response regulator, CheY-P. Because flagellar motors are ultrasensitive, this should change the fraction of time that motors spin clockwise, the CWbias. However, early work failed to show changes in CWbias when ramps were shallow. This was explained by a model for motor remodeling that predicted plateaus in plots of CWbias versus [CheY-P]. We looked for these plateaus by examining distributions of CWbias in populations of cells with different mean [CheY-P]. We did not find such plateaus. Hence, we repeated the work on shallow ramps and found that motors did indeed respond. These responses were quantitatively described by combining motor remodeling with ultrasensitivity in a model that exhibited high sensitivities over a wide dynamic range.

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