Howard C. Berg

Physics of chemoreception.

Statistical fluctuations limit the precision with which a microorganism can, in a given time T, determine the concentration of a chemoattractant in the surrounding medium. The best a cell can do is to monitor continually the state of occupation of receptors distributed over its surface. For nearly optimum performance only a small fraction of the surface need be specifically adsorbing. The probability that a molecule that has collided with the cell will find a receptor is Ns/(Ns + pi a), if N receptors, each with a binding site of radius s, are evenly distributed over a cell of radius a. There is ample room for many indenpendent systems of specific receptors. The adsorption rate for molecules of moderate size cannot be significantly enhanced by motion of the cell or by stirring of the medium by the cell. The least fractional error attainable in the determination of a concentration c is approximately (TcaD) - 1/2, where D is diffusion constant of the attractant. The number of specific receptors needed to attain such precision is about a/s. Data on bacteriophage absorption, bacterial chemotaxis, and chemotaxis in a cellular slime mold are evaluated. The chemotactic sensitivity of Escherichia coli approaches that of the cell of optimum design.

Real-Time Imaging of Fluorescent Flagellar Filaments

Bacteria swim by rotating flagellar filaments that are several micrometers long, but only about 20 nm in diameter. The filaments can exist in different polymorphic forms, having distinct values of curvature and twist. Rotation rates are on the order of 100 Hz. In the past, the motion of individual filaments has been visualized by dark-field or differential-interference-contrast microscopy, methods hampered by intense scattering from the cell body or shallow depth of field, respectively. We have found a simple procedure for fluorescently labeling cells and filaments that allows recording their motion in real time with an inexpensive video camera and an ordinary fluorescence microscope with mercury-arc or strobed laser illumination. We report our initial findings with cells of Escherichia coli. Tumbles (events that enable swimming cells to alter course) are remarkably varied. Not every filament on a cell needs to change its direction of rotation: different filaments can change directions at different times, and a tumble can result from the change in direction of only one. Polymorphic transformations tend to occur in the sequence normal, semicoiled, curly 1, with changes in the direction of movement of the cell body correlated with transformations to the semicoiled form.

Direct observation of extension and retraction of type IV pili.

Type IV pili are thin filaments that extend from the poles of a diverse group of bacteria, enabling them to move at speeds of a few tenths of a micrometer per second. They are required for twitching motility, e.g., in Pseudomonas aeruginosa and Neisseria gonorrhoeae, and for social gliding motility in Myxococcus xanthus. Here we report direct observation of extension and retraction of type IV pili in P. aeruginosa. Cells without flagellar filaments were labeled with an amino-specific Cy3 fluorescent dye and were visualized on a quartz slide by total internal reflection microscopy. When pili were attached to a cell and their distal ends were free, they extended or retracted at rates of about 0.5 μm s−1 (29°C). They also flexed by Brownian motion, exhibiting a persistence length of about 5 μm. Frequently, the distal tip of a filament adsorbed to the substratum and the filament was pulled taut. From the absence of lateral deflections of such filaments, we estimate tensions of at least 10 pN. Occasionally, cell bodies came free and were pulled forward by pilus retraction. Thus, type IV pili are linear actuators that extend, attach at their distal tips, exert substantial force, and retract.

Receptor sensitivity in bacterial chemotaxis

Chemoreceptors in Escherichia coli are coupled to the flagella by a labile phosphorylated intermediate, CheY∼P. Its activity can be inferred from the rotational bias of flagellar motors, but motor response is stochastic and limited to a narrow physiological range. Here we use fluorescence resonance energy transfer to monitor interactions of CheY∼P with its phosphatase, CheZ, that reveal changes in the activity of the receptor kinase, CheA, resulting from the addition of attractants or repellents. Analyses of cheR and/or cheB mutants, defective in receptor methylation/demethylation, show that response sensitivity depends on the activity of CheB and the level of receptor modification. In cheRcheB mutants, the concentration of attractant that generates a half-maximal response is equal to the dissociation constant of the receptor. In wild-type cells, it is 35 times smaller. This amplification, together with the ultrasensitivity of the flagellar motor, explains previous observations of high chemotactic gain.

Functional interactions between receptors in bacterial chemotaxis

Bacterial chemotaxis is a model system for signal transduction, noted for its relative simplicity, high sensitivity, wide dynamic range and robustness. Changes in ligand concentrations are sensed by a protein assembly consisting of transmembrane receptors, a coupling protein (CheW) and a histidine kinase (CheA). In Escherichia coli, these components are organized at the cell poles in tight clusters that contain several thousand copies of each protein. Here we studied the effects of variation in the composition of clusters on the activity of the kinase and its sensitivity to attractant stimuli, monitoring responses in vivo using fluorescence resonance energy transfer. Our results indicate that assemblies of bacterial chemoreceptors work in a highly cooperative manner, mimicking the behaviour of allosteric proteins. Conditions that favour steep responses to attractants in mutants with homogeneous receptor populations also enhance the sensitivity of the response in wild-type cells. This is consistent with a number of models that assume long-range cooperative interactions between receptors as a general mechanism for signal integration and amplification.

Localization of components of the chemotaxis machinery of Escherichia coli using fluorescent protein fusions

We prepared fusions of yellow fluorescent protein [the YFP variant of green fluorescent protein (GFP)] with the cytoplasmic chemotaxis proteins CheY, CheZ and CheA and the flagellar motor protein FliM, and studied their localization in wild‐type and mutant cells of Escherichia coli. All but the CheA fusions were functional. The cytoplasmic proteins CheY, CheZ and CheA tended to cluster at the cell poles in a manner similar to that observed earlier for methyl‐accepting chemotaxis proteins (MCPs), but only if MCPs were present. Co‐localization of CheY and CheZ with MCPs was CheA dependent, and co‐localization of CheA with MCPs was CheW dependent, as expected. Co‐localization with MCPs was confirmed by immunofluorescence using an anti‐MCP primary antibody. The motor protein FliM appeared as discrete spots on the sides of the cell. These were seen in wild‐type cells and in a fliN mutant, but not in flhC or fliG mutants. Co‐localization with flagellar structures was confirmed by immunofluorescence using an antihook primary antibody. Surprisingly, we did not observe co‐localization of CheY with motors, even under conditions in which cells tumbled.

Hydrodynamic attraction of swimming microorganisms by surfaces

Cells swimming in confined environments are attracted by surfaces. We measure the steady-state distribution of smooth-swimming bacteria (Escherichia coli) between two glass plates. In agreement with earlier studies, we find a strong increase of the cell concentration at the boundaries. We demonstrate theoretically that hydrodynamic interactions of the swimming cells with solid surfaces lead to their reorientation in the direction parallel to the surfaces, as well as their attraction by the closest wall. A model is derived for the steady-state distribution of swimming cells, which compares favorably with our measurements. We exploit our data to estimate the flagellar propulsive force in swimming E. coli.

Motile Behavior of Bacteria

Escherichia coli is a single‐celled organism that lives in your gut. It is equipped with a set of rotary motors only 45 nm in diameter. Each motor drives a long, thin, helical filament that extends several cell body lengths out into the external medium. The assemblage of motor and filament is called a flagellum. The concerted motion of several flagella enables a cell to swim. A cell can move toward regions that it deems more favorable by measuring changes in the concentrations of certain chemicals in its environment (mostly nutrients), deciding whether life is getting better or worse, and then modulating the direction of rotation of its flagella. Thus, in addition to rotary engines and propellers, E. colis standard accessories include particle counters, rate meters, and gear boxes. This microorganism is a nanotechnologists dream. I will discuss the features that make it so, from the perspectives of several scientific disciplines: anatomy, genetics, chemistry, and physics.

Escherichia coli swim on the right-hand side.

The motion of peritrichously flagellated bacteria close to surfaces is relevant to understanding the early stages of biofilm formation and of pathogenic infection. This motion differs from the random-walk trajectories of cells in free solution. Individual Escherichia coli cells swim in clockwise, circular trajectories near planar glass surfaces. On a semi-solid agar substrate, cells differentiate into an elongated, hyperflagellated phenotype and migrate cooperatively over the surface, a phenomenon called swarming. We have developed a technique for observing isolated E. coli swarmer cells moving on an agar substrate and confined in shallow, oxidized poly(dimethylsiloxane) (PDMS) microchannels. Here we show that cells in these microchannels preferentially ‘drive on the right’, swimming preferentially along the right wall of the microchannel (viewed from behind the moving cell, with the agar on the bottom). We propose that when cells are confined between two interfaces—one an agar gel and the second PDMS—they swim closer to the agar surface than to the PDMS surface (and for much longer periods of time), leading to the preferential movement on the right of the microchannel. Thus, the choice of materials guides the motion of cells in microchannels.

Sulfanilic acid diazonium salt: A label for the outside of the human erythrocyte membrane

Abstract 1. 1. The diazonium salt of [ 35 S]sulfanilic acid can be used as a label for outer components of the human erythrocyte membrane; the reagent does not penetrate intact cells. 2. 2. Modified cells become permeable to Na + and K + but not to water-soluble nonelectrolytes. They eventually lyse in isotonic buffer. 3. 3. About 20% of the label bound to intact cells can be recovered in an ethanol-ether membrane extract. Phospholipase D (cabbage) changes the way in which it partitions between ether and water. 4. 4. If the residue left after ethanol-ether extraction is dissolved in 3% sodium dodecyl sulfate and sized by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate, a complex but reproducible pattern is observed on staining with Coomassie brilliant blue. The most intensely labeled material has a molecular weight of about 140 000. Some peaks are free of label. 5. 5. Similar patterns are obtained if intact membranes are dissolved in 3% sodium dodecyl sulfate; sodium dodecyl sulfate dissociates the protein and lipid as effectively as ethanol-ether. 6. 6. If the residue left after ethanol-ether extraction is exposed first to 0.8 M NaCl, a medium in which it is largely insoluble, much of the protein shifts from high to low molecular weight. The most intensely labeled material does not. The membrane contains protein complexes which can be dissociated by sodium dodecyl sulfate after exposure to salt. The dissociation is less extensive when intact membranes are exposed to salt.