RESUMEN
Bacterial swarming is a type of motility characterized by a rapid and collective migration of bacteria on surfaces. Most swarming species form densely packed dynamic clusters in the form of whirls and jets, in which hundreds of rod-shaped rigid cells move in circular and straight patterns, respectively. Recent studies have suggested that short-range steric interactions may dominate hydrodynamic interactions and that geometrical factors, such as a cell's aspect ratio, play an important role in bacterial swarming. Typically, the aspect ratio for most swarming species is only up to 5, and a detailed understanding of the role of much larger aspect ratios remains an open challenge. Here we study the dynamics of Paenibacillus dendritiformis C morphotype, a very long, hyperflagellated, straight (rigid), rod-shaped bacterium with an aspect ratio of ~20. We find that instead of swarming in whirls and jets as observed in most species, including the shorter T morphotype of P. dendritiformis, the C morphotype moves in densely packed straight but thin long lines. Within these lines, all bacteria show periodic reversals, with a typical reversal time of 20 s, which is independent of their neighbors, the initial nutrient level, agar rigidity, surfactant addition, humidity level, temperature, nutrient chemotaxis, oxygen level, illumination intensity or gradient, and cell length. The evolutionary advantage of this unique back-and-forth surface translocation remains unclear.
Asunto(s)
Locomoción , Paenibacillus/fisiología , Medios de Cultivo/química , Flagelos/fisiología , Flagelos/ultraestructura , Microscopía Electrónica de Transmisión , Paenibacillus/ultraestructuraRESUMEN
Flocking birds, fish schools, and insect swarms are familiar examples of collective motion that plays a role in a range of problems, such as spreading of diseases. Models have provided a qualitative understanding of the collective motion, but progress has been hindered by the lack of detailed experimental data. Here we report simultaneous measurements of the positions, velocities, and orientations as a function of time for up to a thousand wild-type Bacillus subtilis bacteria in a colony. The bacteria spontaneously form closely packed dynamic clusters within which they move cooperatively. The number of bacteria in a cluster exhibits a power-law distribution truncated by an exponential tail. The probability of finding clusters with large numbers of bacteria grows markedly as the bacterial density increases. The number of bacteria per unit area exhibits fluctuations far larger than those for populations in thermal equilibrium. Such "giant number fluctuations" have been found in models and in experiments on inert systems but not observed previously in a biological system. Our results demonstrate that bacteria are an excellent system to study the general phenomenon of collective motion.
Asunto(s)
Bacillus subtilis/citología , Movimiento (Física) , Análisis por Conglomerados , Viabilidad Microbiana , Modelos BiológicosRESUMEN
Sibling Paenibacillus dendritiformis bacterial colonies grown on low-nutrient agar medium mutually inhibit growth through secretion of a lethal factor. Analysis of secretions reveals the presence of subtilisin (a protease) and a 12 kDa protein, termed sibling lethal factor (Slf). Purified subtilisin promotes the growth and expansion of P. dendritiformis colonies, whereas Slf is lethal and lyses P. dendritiformis cells in culture. Slf is encoded by a gene belonging to a large family of bacterial genes of unknown function, and the gene is predicted to encode a protein of approximately 20 kDa, termed dendritiformis sibling bacteriocin. The 20 kDa recombinant protein was produced and found to be inactive, but exposure to subtilisin resulted in cleavage to the active, 12 kDa form. The experimental results, combined with mathematical modeling, show that subtilisin serves to regulate growth of the colony. Below a threshold concentration, subtilisin promotes colony growth and expansion. However, once it exceeds a threshold, as occurs at the interface between competing colonies, Slf is then secreted into the medium to rapidly reduce cell density by lysis of the bacterial cells. The presence of genes encoding homologs of dendritiformis sibling bacteriocin in other bacterial species suggests that this mechanism for self-regulation of colony growth might not be limited to P. dendritiformis.
Asunto(s)
Proteínas Bacterianas/metabolismo , Paenibacillus/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Viabilidad Microbiana , Datos de Secuencia Molecular , Peso Molecular , Paenibacillus/química , Paenibacillus/crecimiento & desarrollo , Subtilisina/metabolismoRESUMEN
Bacteria can secrete a wide array of antibacterial compounds when competing with other bacteria for the same resources. Some of these compounds, such as bacteriocins, can affect bacteria of similar or closely related strains. In some cases, these secretions have been found to kill sibling cells that belong to the same colony. Here, we present experimental observations of competition between 2 sibling colonies of Paenibacillus dendritiformis grown on a low-nutrient agar gel. We find that neighboring colonies (growing from droplet inoculation) mutually inhibit growth through secretions that become lethal if the level exceeds a well-defined threshold. In contrast, within a single colony developing from a droplet inoculation, no growth inhibition is observed. However, growth inhibition and cell death are observed if material extracted from the agar between 2 growing colonies is introduced outside a growing single colony. To interpret the observations, we devised a simple mathematical model for the secretion of an antibacterial compound. Simulations of this model illustrate how secretions from neighboring colonies can be deadly, whereas secretions from a single colony growing from a droplet are not.
Asunto(s)
Bacterias/citología , Fenómenos Fisiológicos Bacterianos , Agar , Antibacterianos , Bacterias/química , Bacteriocinas , Comunicación Celular , Alimentos , Modelos BiológicosRESUMEN
Most research on growing bacterial colonies on agar plates has concerned the effect of genetic or morphotype variation. Some studies have indicated that there is a correlation between microscopic bacterial motion and macroscopic colonial expansion, especially for swarming strains, but no measurements have been obtained for a single strain to relate the microscopic scale to the macroscopic scale. We examined here a single strain (Paenibacillus dendritiformis type T; tip splitting) to determine both the macroscopic growth of colonies and the microscopic bacterial motion within the colonies. Our multiscale measurements for a variety of growth conditions revealed that motion on the microscopic scale and colonial growth are largely independent. Instead, the growth of the colony is strongly affected by the availability of a surfactant that reduces surface tension.
Asunto(s)
Bacilos Grampositivos Formadores de Endosporas/crecimiento & desarrollo , Bacilos Grampositivos Formadores de Endosporas/fisiología , Tensoactivos/metabolismo , Tensoactivos/farmacología , Agar , Técnicas Bacteriológicas , Recuento de Colonia Microbiana , Medios de Cultivo , Bacilos Grampositivos Formadores de Endosporas/efectos de los fármacos , Microscopía de Contraste de Fase , Movimiento/efectos de los fármacosRESUMEN
To probe the dynamics and size of lipid rafts in the membrane of living cells, the local diffusion of single membrane proteins was measured. A laser trap was used to confine the motion of a bead bound to a raft protein to a small area (diam < or = 100 nm) and to measure its local diffusion by high resolution single particle tracking. Using protein constructs with identical ectodomains and different membrane regions and vice versa, we demonstrate that this method provides the viscous damping of the membrane domain in the lipid bilayer. When glycosylphosphatidylinositol (GPI) -anchored and transmembrane proteins are raft-associated, their diffusion becomes independent of the type of membrane anchor and is significantly reduced compared with that of nonraft transmembrane proteins. Cholesterol depletion accelerates the diffusion of raft-associated proteins for transmembrane raft proteins to the level of transmembrane nonraft proteins and for GPI-anchored proteins even further. Raft-associated GPI-anchored proteins were never observed to dissociate from the raft within the measurement intervals of up to 10 min. The measurements agree with lipid rafts being cholesterol-stabilized complexes of 26 +/- 13 nm in size diffusing as one entity for minutes.
Asunto(s)
Membrana Celular/metabolismo , Colesterol/metabolismo , Esfingolípidos/metabolismo , Fosfatasa Alcalina , Animales , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Línea Celular , Colesterol/química , Cricetinae , Difusión , Técnica del Anticuerpo Fluorescente , Colorantes Fluorescentes , Proteínas Ligadas a GPI , Glicosilfosfatidilinositoles/metabolismo , Hemaglutininas Virales/biosíntesis , Hemaglutininas Virales/genética , Isoenzimas/biosíntesis , Rayos Láser , Membrana Dobles de Lípidos/metabolismo , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Proteínas de la Membrana/metabolismo , Microesferas , Tamaño de la Partícula , Esfingolípidos/química , Transfección , ViscosidadRESUMEN
The recognition mechanisms and dissociation pathways of the avidin-biotin complex and of actin monomers in actin filaments were investigated. The unbinding forces of discrete complexes of avidin or streptavidin with biotin analogs are proportional to the enthalpy change of the complex formation but independent of changes in the free energy. This result indicates that the unbinding process is adiabatic and that entropic changes occur after unbinding. On the basis of the measured forces and binding energies, an effective rupture length of 9.5 +/- 1 angstroms was calculated for all biotin-avidin pairs and approximately 1 to 3 angstroms for the actin monomer-monomer interaction. A model for the correlation among binding forces, intermolecular potential, and molecular function is proposed.
Asunto(s)
Avidina/química , Biotina/química , Ligandos , Receptores de Droga/química , Avidina/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Biotina/análogos & derivados , Biotina/metabolismo , Concentración de Iones de Hidrógeno , Modelos Químicos , Estreptavidina , TermodinámicaRESUMEN
The adhesion force between the tip of an atomic force microscope cantilever derivatized with avidin and agarose beads functionalized with biotin, desthiobiotin, or iminobiotin was measured. Under conditions that allowed only a limited number of molecular pairs to interact, the force required to separate tip and bead was found to be quantized in integer multiples of 160 +/- 20 piconewtons for biotin and 85 +/- 15 piconewtons for iminobiotin. The measured force quanta are interpreted as the unbinding forces of individual molecular pairs.
Asunto(s)
Avidina/química , Biotina/química , Receptores de Superficie Celular/química , Adhesividad , Biotina/análogos & derivados , Ligandos , Microscopía/métodos , Microesferas , SefarosaRESUMEN
A quadrant photodiode placed in the back-focal plane of the microscope of a laser trap provides a high-resolution position sensor. We show that in addition to the lateral displacement of a trapped sphere, its axial position can be measured by the ratio of the intensity of scattered laser light to the total amount of the light reaching the detector. The addition of the axial information offers true three-dimensional position detection in solution, creating, together with a position control, a photonic force microscope with nanometer spatial and microsecond temporal resolution. The measured position signals are explained as interference of the unscattered trapping laser beam with the laser light scattered by the trapped bead. Our model explains experimental data for trapped particles in the Rayleigh regime (radius a <0.2lambda) for displacements up to the focal dimensions. The cross-talk between the signals in the three directions is explained and it is shown that this cross-talk can be neglected for lateral displacements smaller than 75 nm and axial displacements below 150 nm. The advantages of three-dimensional single-particle tracking over conventional video-tracking are shown through the example of the diffusion of the GPI-anchored membrane protein Thy1.1 on a neurite.
Asunto(s)
Proteínas de la Membrana/metabolismo , Microscopía/instrumentación , Microscopía/métodos , Neuritas/metabolismo , Antígenos Thy-1/metabolismo , Animales , Transporte Biológico , Difusión , Hipocampo/embriología , Rayos Láser , Micromanipulación , Óptica y Fotónica , Tamaño de la Partícula , Fotones , Ratas , Dispersión de RadiaciónRESUMEN
UNLABELLED: Natural habitats vary in available nutrients and room for bacteria to grow, but successful colonization can lead to overcrowding and stress. Here we show that competing sibling colonies of Paenibacillus dendritiformis bacteria survive overcrowding by switching between two distinct vegetative phenotypes, motile rods and immotile cocci. Growing colonies of the rod-shaped bacteria produce a toxic protein, Slf, which kills cells of encroaching sibling colonies. However, sublethal concentrations of Slf induce some of the rods to switch to Slf-resistant cocci, which have distinct metabolic and resistance profiles, including resistance to cell wall antibiotics. Unlike dormant spores of P. dendritiformis, the cocci replicate. If cocci encounter conditions that favor rods, they secrete a signaling molecule that induces a switch to rods. Thus, in contrast to persister cells, P. dendritiformis bacteria adapt to changing environmental conditions by inducible and reversible phenotypic switching. IMPORTANCE: In favorable environments, species may face space and nutrient limits due to overcrowding. Bacteria provide an excellent model for analyzing principles underlying overcrowding and regulation of density in nature, since their population dynamics can be easily and accurately assessed under controlled conditions. We describe a newly discovered mechanism for survival of a bacterial population during overcrowding. When competing with sibling colonies, Paenibacillus dendritiformis produces a lethal protein (Slf) that kills cells at the interface of encroaching colonies. Slf also induces a small proportion of the cells to switch from motile, rod-shaped cells to nonmotile, Slf-resistant, vegetative cocci. When crowding is reduced and nutrients are no longer limiting, the bacteria produce a signal that induces cocci to switch back to motile rods, allowing the population to spread. Genes encoding components of this phenotypic switching pathway are widespread among bacterial species, suggesting that this survival mechanism is not unique to P. dendritiformis.
Asunto(s)
Locomoción , Viabilidad Microbiana , Paenibacillus/citología , Paenibacillus/fisiología , Estrés Fisiológico , Antibacterianos/metabolismo , Toxinas Bacterianas/biosíntesis , Toxinas Bacterianas/toxicidad , Regulación Bacteriana de la Expresión Génica , Paenibacillus/efectos de los fármacos , Paenibacillus/crecimiento & desarrollo , FenotipoAsunto(s)
Cinesinas/fisiología , Microscopía de Fuerza Atómica/métodos , Microtúbulos/ultraestructura , Proteínas Motoras Moleculares , Animales , Calibración , Embrión de Pollo , Procesamiento de Imagen Asistido por Computador , Micromanipulación/instrumentación , Microesferas , Microtúbulos/efectos de los fármacos , Microtúbulos/fisiología , Proteínas del Tejido Nervioso/fisiología , Paclitaxel/farmacologíaRESUMEN
The thermal position fluctuations of a single micron-sized sphere immersed in a fluid were recorded by optical trapping interferometry with nanometer spatial and microsecond temporal resolution. We find, in accord with the theory of Brownian motion including hydrodynamic memory effects, that the transition from ballistic to diffusive motion is delayed to significantly longer times than predicted by the standard Langevin equation. This delay is a consequence of the inertia of the fluid. On the shortest time scales investigated, the sphere's inertia has a small, but measurable, effect.
RESUMEN
We report herein measurements on a novel type of supported lipid films, which we call painted supported membranes (PSM). These membranes are formed in a self-assembly process on alkylated gold films from an organic solution. The formation process was investigated with surface plasmon resonance microscopy. The optical and electrical properties of the films were determined for various types of lipids and as a function of temperature by means of cyclic voltammetry and potential relaxation after charge injection. We could show that these films exhibit an extraordinarily high specific resistivity which, depending on the lipid, may be as high as 10(9) ohm/cm(2). We could also show that due to this low conductivity, an electrical polarization across the PSM relaxes with characteristic time constants of up to 20 min. The electrical properties together with their high mechanical stability and accessibility to surface sensitive techniques make these films well suitable model membranes for optical and electrical investigations. Examples for such applications are given in the subsequent article by Seifert et al.
RESUMEN
We show that the optical trapping of dielectric particles by a single focused beam in front of a weakly reflective surface is considerably affected by interference of the incident and reflected beams, which creates a standing-wave component of the total field. We use the two-photon-excited fluorescence from a trapped dyed probe to detect changes in the distance between the trapped beam focus as the focus approaches the reflective surface. This procedure enables us to determine the relative strengths of the single-beam and the standing-wave trapping forces. We demonstrate that, even for reflection from a glass-water interface, standing-wave trapping dominates, as far as 5 mum from the surface.
RESUMEN
A new scanning probe microscope, the photonic force microscope (PFM), based on optical tweezers and two-photon absorption processes for biological applications is described. Optical tweezers are used to trap a fluorescent latex bead with a diameter of 200 nm in an aqueous solution in all three dimensions. The fluorescent dye is chosen to fulfill the two-photon absorption criterion for the 1064-nm line of a Nd:YVO4 laser. The intensity of the fluorescence emission is utilized as a very sensitive position sensor along the optical axis. Two-dimensional images are formed by laterally scanning the trapped latex bead across biological samples while recording the two-photon-induced fluorescences intensity. A scanning probe image of the outer surface of a small neurite from a cultured rat hippocampal neuron is shown, which is hardly visible under differential interference contrast microscopy. The lateral resolution is given by the bead diameter; the axial resolution is 40 nm. Under the experimental conditions the maximal imaging force applied by the probe is below 5 pN.