RESUMEN
Microscopy has served as a fundamental tool for insight and discovery in plant-microbe interactions for centuries. From classical light and electron microscopy to corresponding specialized methods for sample preparation and cellular contrasting agents, these approaches have become routine components in the toolkit of plant and microbiology scientists alike to visualize, probe and understand the nature of host-microbe relationships. Over the last three decades, three-dimensional perspectives led by the development of electron tomography, and especially, confocal techniques continue to provide remarkable clarity and spatial detail of tissue and cellular phenomena. Confocal and electron microscopy provide novel revelations that are now commonplace in medium and large institutions. However, many other cutting-edge technologies and sample preparation workflows are relatively unexploited yet offer tremendous potential for unprecedented advancement in our understanding of the inner workings of pathogenic, beneficial, and symbiotic plant-microbe interactions. Here, we highlight key applications, benefits, and challenges of contemporary advanced imaging platforms for plant-microbe systems with special emphasis on several recently developed approaches, such as light-sheet, single molecule, super-resolution, and adaptive optics microscopy, as well as ambient and cryo-volume electron microscopy, X-ray microscopy, and cryo-electron tomography. Furthermore, the potential for complementary sample preparation methodologies, such as optical clearing, expansion microscopy, and multiplex imaging, will be reviewed. Our ultimate goal is to stimulate awareness of these powerful cutting-edge technologies and facilitate their appropriate application and adoption to solve important and unresolved biological questions in the field. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.
Asunto(s)
Microscopía por Crioelectrón , Interacciones Microbiota-Huesped , Plantas , Microscopía por Crioelectrón/métodos , Interacciones Microbiota-Huesped/fisiología , Plantas/microbiologíaRESUMEN
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.
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 , TorqueRESUMEN
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 SuperficieRESUMEN
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.
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Fenómenos Fisiológicos Bacterianos , Movimiento/fisiología , Adhesinas Bacterianas/fisiología , Animales , Bacterias , Microscopía por Crioelectrón/métodos , Fimbrias Bacterianas/fisiologíaRESUMEN
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.
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Proteínas Bacterianas , Flagelos , Bacterias/metabolismo , Proteínas Bacterianas/química , Microscopía por Crioelectrón/métodos , Flagelos/química , Flagelos/metabolismo , Flagelos/ultraestructura , TorqueRESUMEN
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).
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Flagelos/química , Mecanotransducción Celular , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Escherichia coli , Flagelos/metabolismo , Modelos Teóricos , RotaciónRESUMEN
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.
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Proteínas Bacterianas/ultraestructura , Flagelos/ultraestructura , Bacterias/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Microscopía por Crioelectrón/métodos , Flagelos/metabolismo , Conformación Proteica , TorqueRESUMEN
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.
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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 , RotaciónRESUMEN
One of the major factors limiting biomass productivity in algae is the low thermodynamic efficiency of photosynthesis. The greatest thermodynamic inefficiencies in photosynthesis occur during the conversion of light into chemical energy. At full sunlight the light-harvesting antenna captures photons at a rate nearly 10 times faster than the rate-limiting step in photosynthetic electron transport. Excess captured energy is dissipated by non-productive pathways including the production of reactive oxygen species. Substantial improvements in photosynthetic efficiency have been achieved by reducing the optical cross-section of the light-harvesting antenna by selectively reducing chlorophyll b levels and peripheral light-harvesting complex subunits. Smaller light-harvesting antenna, however, may not exhibit optimal photosynthetic performance in low or fluctuating light environments. We describe a translational control system to dynamically adjust light-harvesting antenna sizes for enhanced photosynthetic performance. By expressing a chlorophyllide a oxygenase (CAO) gene having a 5' mRNA extension encoding a Nab1 translational repressor binding site in a CAO knockout line it was possible to continuously alter chlorophyll b levels and correspondingly light-harvesting antenna sizes by light-activated Nab1 repression of CAO expression as a function of growth light intensity. Significantly, algae having light-regulated antenna sizes had substantially higher photosynthetic rates and two-fold greater biomass productivity than the parental wild-type strains as well as near wild-type ability to carry out state transitions and non-photochemical quenching. These results have broad implications for enhanced algae and plant biomass productivity.
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Chlorophyta/metabolismo , Complejos de Proteína Captadores de Luz/fisiología , Fotosíntesis , Biomasa , Clorofila/metabolismo , Chlorophyta/crecimiento & desarrollo , Chlorophyta/fisiología , Luz , Complejos de Proteína Captadores de Luz/metabolismo , Fotosíntesis/fisiología , Plantas Modificadas GenéticamenteRESUMEN
Oil palm breeding involves crossing dura and pisifera palms to produce tenera progeny with greatly improved oil yield. Oil yield is controlled by variant alleles of a type II MADS-box gene, SHELL, that impact the presence and thickness of the endocarp, or shell, surrounding the fruit kernel. We identified six novel SHELL alleles in noncommercial African germplasm populations from the Malaysian Palm Oil Board. These populations provide extensive diversity to harness genetic, mechanistic and phenotypic variation associated with oil yield in a globally critical crop. We investigated phenotypes in heteroallelic combinations, as well as SHELL heterodimerization and subcellular localization by yeast two-hybrid, bimolecular fluorescence complementation and gene expression analyses. Four novel SHELL alleles were associated with fruit form phenotype. Candidate heterodimerization partners were identified, and interactions with EgSEP3 and subcellular localization were SHELL allele-specific. Our findings reveal allele-specific mechanisms by which variant SHELL alleles impact yield, as well as speculative insights into the potential role of SHELL in single-gene oil yield heterosis. Future field trials for combinability and introgression may further optimize yield and improve sustainability.
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Arecaceae , Fitomejoramiento , Alelos , Arecaceae/genética , Aceite de Palma , FenotipoRESUMEN
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.
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Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Flagelos/metabolismo , Sustancias Macromoleculares/metabolismo , Proteínas Motoras Moleculares/metabolismo , TorqueRESUMEN
Cervical necrotising fasciitis (NF) is an aggressive polymicrobial infection of the subcutaneous tissues in the head and neck. We present a case of a healthy 19-year-old man who developed cervical and upper mediastinal NF after an initial presentation of infectious mononucleosis (IM). He was treated with broad-spectrum antibiotics in addition to incision and drainage of an anterior neck and upper mediastinal abscess. He progressed favourably after ten days of hospitalisation and was discharged home on intravenous antibiotics. This is a unique case of cervical NF as a sequelae of IM in a previously healthy paediatric patient.
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Fascitis Necrotizante/virología , Herpesvirus Humano 4 , Mononucleosis Infecciosa/microbiología , Cuello/microbiología , Humanos , Mononucleosis Infecciosa/complicaciones , Masculino , Adulto JovenRESUMEN
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.
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Capnocytophaga/fisiología , Consorcios Microbianos/fisiología , Microbiota/fisiología , Boca/microbiología , HumanosRESUMEN
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.
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Bacterias/citología , Flagelos/ultraestructura , Colorantes Fluorescentes/metabolismo , Bacterias/metabolismo , Flagelos/metabolismo , Procesamiento de Imagen Asistido por Computador , Microscopía Fluorescente/métodos , Grabación en VideoRESUMEN
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.
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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 ProteicaRESUMEN
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.
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Bacterias , Flagelos , Modelos Biológicos , Fenómenos Fisiológicos Bacterianos , Simulación por Computador , Movimiento , Rotación , Torsión Mecánica , Sustancias Viscoelásticas , ViscosidadRESUMEN
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.
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Quimiotaxis/fisiología , Escherichia coli/fisiología , Flagelos/fisiología , Transducción de Señal/fisiologíaRESUMEN
Flavobacterium johnsoniae, a rod-shaped bacterium, glides over surfaces at speeds of â¼2 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.