Response of Escherichia coli to novel gradients.
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Chemotaxis↗
Biomedical subjects
H C Berg
Publications and source records attributed to H C Berg.
Mutations in the MotA protein of Escherichia coli reveal domains critical for proton conduction.
The MotA protein of Escherichia coli is an essential component of the torque-generating units that drive the flagellar rotary motor. A variety of evidence indicates that MotA is involved in transmembrane proton conduction. We have now mapped a number of MotA mutants, focusing primarily on those previously shown to be dominant. Fifty-six mutations (all dominant), each causing severe or complete impairment of function, were sequenced and found to correspond to 31 different alleles. All except two of these encoded amino acid substitutions clustered in four hydrophobic, presumably membrane-spanning segments, that together make up only one-third of the length of the polypeptide chain. In contrast, eight mutations (5 dominant), each causing only slight impairment of function (slow alleles), were sequenced and found to specify amino acid substitutions in three hydrophilic domains. The clustering of the mutations provides independent support for the suggestion that MotA is a transmembrane proton channel and places significant constraints on models for the molecular mechanism of ion conduction.
Alleles↗
Selection of motile nonchemotactic mutants of Escherichia coli by field-flow fractionation.
We have developed a chromatographic method for isolating bacterial cells that are motile but nonchemotactic. Separation of strains of different phenotype occurs along a thin horizontal channel between two stirred chambers, the lower one containing a chemical attractant. The channel is bounded above and below by rigid filters, permeable to the attractant but not to the bacteria. The lower part of the channel is occupied by a porous plate comprising a vertical array of capillary tubes. An aliquot of cells is injected at one end of the channel and eluted by continuous flow of cell-free medium. Fluid leaving the other end of the channel is collected in a fraction collector. Cells that respond to the gradient swim to the bottom of the channel where they are retarded by the capillary array. Nonmotile cells sink to the bottom and are trapped in a similar manner. Motile cells that fail to respond to the gradient diffuse across the full height of the channel and, thus, travel through the apparatus at the average velocity of the eluent. When mixed with wild-type cells at a ratio of 1:1000 and subjected to an aspartate gradient, aspartate-blind cells were recovered quantitatively. The enrichment was approximately 200 to 1. The wild-type cells that survived the selection had a poorly motile phenotype.
Cell Separation↗
Complex patterns formed by motile cells of Escherichia coli.
When chemotactic strains of the bacterium Escherichia coli are inoculated on semi-solid agar containing mixtures of amino acids or sugars, the cells swarm outwards in a series of concentric rings: they respond to spatial gradients of attractants generated by uptake and catabolism. Cells also drift up gradients generated artificially, for example by diffusion from the tip of a capillary tube or by mixing. Here we describe conditions under which cells aggregate in response to gradients of attractant which they excrete themselves. When cells are grown in semi-solid agar on intermediates of the tricarboxylic acid cycle, they form symmetrical arrays of spots or stripes that arise sequentially. When cells in a thin layer of liquid culture are exposed to these compounds, spots appear synchronously, more randomly arrayed. In either case, the patterns are stationary. The attractant is a chemical sensed by the aspartate receptor. Its excretion can be triggered by oxidative stress. As oxygen is limiting at high cell densities, aggregation might serve as a mechanism for collective defence.
Aspartic Acid↗
Bacterial motility: handedness and symmetry.
Many bacteria swim by rotating thin helical filaments that extend into the external medium, as with common bacteria, or run beneath the outer membrane, as with spirochetes. Each filament is driven at its base by a motor that turns alternately clockwise and counterclockwise. The motor-filament complex is called a flagellum. Other kinds of bacteria glide, but their organelles of locomotion are not known. Since bacteria are microscopic and live in an aqueous environment, they swim at low Reynolds' number; cyclic motion works (e.g. rotation of a helix) but reciprocal motion does not (e.g. stroking of a singly hinged oar). By measuring concentrations of certain chemicals as they move through their environment, making temporal comparisons and modulating the direction of flagellar rotation, bacteria accumulate in regions that they find more favourable. Studies of bacterial chemotaxis are highly advanced, particularly for the peritrichously flagellated species Escherichia coli. A great deal is known about chemoreception, receptor-flagellar coupling and adaptation. Recently it has been found that E. coli can aggregate in response to signals generated by the cells themselves. Complex patterns form with remarkable symmetries.
Bacterial Physiological Phenomena↗
Compliance of bacterial polyhooks measured with optical tweezers.
In earlier work, a single-beam gradient force optical trap ("optical tweezers") was used to measure the torsional compliance of flagella in wild-type cells of Escherichia coli that had been tethered to glass by a single flagellum. This compliance was nonlinear, exhibiting a torsionally soft phase up to 180 degrees, followed by a torsionally rigid phase for larger angles. Values for the torsional spring constant in the soft phase were substantially less than estimates based on the rigidity determined for isolated flagellar filaments. It was suggested that the soft phase might correspond to wind-up of the flagellar hook, and the rigid phase to wind-up of the stiffer filament. Here, we have measured the torsional compliance of flagella on cells of an E. coli strain that produces abnormally long hooks but no filaments. The small-angle compliance of these cells, as determined from the elastic rebound of the cell body after wind-up and release, was found to be the same as for wild-type cells. This confirms that the small-angle compliance of wild-type cells is dominated by the response of the hook. Hook flexibility is likely to play a useful role in stabilizing the flagellar bundle.
Bacterial Proteins↗
Mutant MotB proteins in Escherichia coli.
The MotB protein of Escherichia coli is an essential component in each of eight torque generators in the flagellar rotary motor. Based on its membrane topology, it has been suggested that MotB might be a linker that fastens the torque-generating machinery to the cell wall. Here, we report the isolation and characterization of a number of motB mutants. As found previously for motA, many alleles of motB were dominant, as expected if MotB is a component of the motor. In other respects, however, the motB mutants differed from the motA mutants. Most of the mutations mapped to a hydrophilic, periplasmic domain of the protein, and nothing comparable to the slow-swimming alleles of motA, which show normal torque when tethered, was found. Some motB mutants retained partial function, but when tethered they produced subnormal torque, indicating that their motors contained only one or two functional torque generators. These results support the hypothesis that MotB is a linker.
Alleles↗
Visualization of bacterial flagella by video-enhanced light microscopy.
We have imaged individual flagellar filaments of Escherichia coli, a motile Streptococcus sp., and Rhizobium meliloti by video-enhanced differential interference-contrast microscopy (Nomarski DIC) and computer-based image processing. This approach has advantages over existing methods in that filaments on living cells can be seen over their entire lengths.
Cell Movement↗
Evidence for interactions between MotA and MotB, torque-generating elements of the flagellar motor of Escherichia coli.
Cells that overexpress MotA (encoded on a plasmid derived from pBR322) grow slowly because of proton leakage. We have traced this defect to the coexpression of a fusion protein consisting of 60 amino acids from the N terminus of MotB and 50 amino acids specified by pBR322. Mutations within the N terminus, known to abolish function when present in full-length MotB, reversed the growth defect. Growth also was normal when MotA was coexpressed with wild-type MotB or with a series of MotB N-terminal fragments.
Amino Acid Sequence↗
The MotA protein of E. coli is a proton-conducting component of the flagellar motor.
A number of mutants of motA, a gene necessary for flagellar rotation in E. coli, were isolated and characterized. Many mutations were dominant, owing to competition between functional and nonfunctional MotA for a limited number of sites on the flagellar motor. A new class of mutant was discovered in which flagellar torque is normal at low speeds but reduced at high speeds. Hydrogen isotope effects on these mutants indicate that MotA catalyzes proton transfer. We confirmed an earlier observation that overproduction of MotA leads to accumulation of the protein in the cytoplasmic membrane and to significant decreases in growth rate. When nonfunctional mutant variants of MotA were overproduced instead, they accumulated in the cytoplasmic membrane, but growth was not impaired. These results also suggest that MotA conducts protons. This was confirmed by measuring the proton permeabilities of vesicles containing wild-type or mutant MotA proteins.
Alleles↗
Chemotaxis of bacteria in glass capillary arrays. Escherichia coli, motility, microchannel plate, and light scattering.
Random and directed motility of bacterial populations were assayed by monitoring the flux of bacteria through a microchannel plate (a porous glass plate comprising a fused array of capillary tubes) separating two identical stirred chambers. Cells, washed free of growth medium by a new filtration method, were added to one chamber at a low density. Their number in the other chamber was determined from the amount of light scattered from a beam of a laser diode and recorded on a strip chart. Diffusion coefficients were computed from fluxes observed in the absence of chemical gradients, and chemotaxis drift velocities were computed from fluxes observed in their presence. Cells migrated through tubes of diam 10 microns more rapidly than through tubes of diam 50 microns, suggesting that the straight segments of their tracks were aligned with the axes of the smaller tubes. Mutants that are motile but nonchemotactic could be selected because they move through the microchannel plate in the face of an adverse gradient. Weak chemotactic responses were assessed from ratios of fluxes observed in paired experiments in which the sign of the gradient of attractant was reversed. Studies were made of wild-type Escherichia coli and mutants that are nonmotile, tumblely, smooth-swimming, aspartate-blind, or defective in methylation and demethylation. Chemotaxis drift velocities for the latter mutants (cheRcheB) were quite small.
Biophysical Phenomena↗
Bacterial microprocessing.
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Adaptation, Physiological↗
Compliance of bacterial flagella measured with optical tweezers.
The development of the gradient force optical particle trap ('optical tweezers') has made it possible to manipulate biological materials using a single beam of laser light. Optical traps can produce forces in the microdyne range on intact cells without causing overt damage: such forces are sufficient to arrest actively swimming bacteria and can overcome torque generated by the flagellar motor of a bacterium tethered to a glass surface by a flagellar filament. By calibrating the trapping force against Stokes' drag and measuring the twist that is sustained by this force, we determined the torsional compliance of flagella in tethered Escherichia coli and a motile Streptococcus. Flagella behaved as linear torsion springs for roughly half a revolution, but became much more rigid when turned beyond this point in either direction.
Escherichia coli↗
Dynamics of a tightly coupled mechanism for flagellar rotation. Bacterial motility, chemiosmotic coupling, protonmotive force.
The bacterial flagellar motor is a molecular engine that couples the flow of protons across the cytoplasmic membrane to rotation of the flagellar filament. We analyze the steady-state behavior of an explicit mechanical model in which a fixed number of protons carries the filament through one revolution. Predictions of this model are compared with experimentally determined relationships between protonmotive force, proton flux, torque, and speed. All such tightly coupled mechanisms produce the same torque when the motor is stalled but vary greatly in their behavior at high speed. The speed at zero load predicted by our model is limited by the rates of association and dissociation of protons at binding sites on the rotor and by the mobility of force generators containing transmembrane channels that interact with these sites. Our analysis suggests that more could be learned about the motor if it were driven by an externally applied torque backwards (at negative speed) or forwards at speeds greater than the zero-load speed.
Bacterial Physiological Phenomena↗
Migration of bacteria in semisolid agar.
We studied the migration through semisolid agar of chemotactic and nonchemotactic cells of Escherichia coli. While swarms of nonchemotactic cells were generally smaller than those of chemotactic cells, they varied markedly in size and in structure. Cells that failed to tumble or that tumbled incessantly formed the smallest swarms. Cells that tumbled at intermediate frequencies formed much larger swarms, even when deleted for many of the genes known to be required for chemotaxis. Surprisingly, the higher the tumble frequency, the larger the swarms. Microscopic examination revealed that tumbles enable cells to back away from obstructions in the agar. Thus, not all cells that swarm effectively need be chemotactic.
Agar↗
Both CheA and CheW are required for reconstitution of chemotactic signaling in Escherichia coli.
If cells of Escherichia coli deleted for genes that specify transducers and all known cytoplasmic chemotaxis proteins are reconstituted with CheA, CheW, and CheY, they spin their flagella alternately clockwise and counterclockwise. If the aspartate receptor also is present, clockwise rotation is suppressed upon addition of aspartate. If either CheA or CheW is absent, the fraction of time that the flagella spin clockwise is reduced and responses to aspartate do not occur.
Bacterial Proteins↗
Restoration of torque in defective flagellar motors.
Paralyzed motors of motA and motB point and deletion mutants of Escherichia coli were repaired by synthesis of wild-type protein. As found earlier with a point mutant of motB, torque was restored in a series of equally spaced steps. The size of the steps was the same for both MotA and MotB. Motors with one torque generator spent more time spinning counterclockwise than did motors with two or more generators. In deletion mutants, stepwise decreases in torque, rare in point mutants, were common. Several cells stopped accelerating after eight steps, suggesting that the maximum complement of torque generators is eight. Each generator appears to contain both MotA and MotB.
Bacterial Proteins↗
Acetyladenylate plays a role in controlling the direction of flagellar rotation.
Cells of Escherichia coli deleted for genes that code for the transducers and all the known cytoplasmic Che proteins except CheY responded reversibly to the addition of acetate by spinning their flagellar motors clockwise. By varying growth conditions and using metabolic inhibitors and mutants deficient in acetate metabolism, this effect was shown to require acetate-CoA synthetase [acetate:CoA ligase (AMP-forming); EC 6.2.1.1], an enzyme that catalyzes the formation of acetyl-CoA from acetate by an acetyladenylate intermediate. A mutant deficient in this enzyme but retaining the chemotaxis genes was deficient for chemotaxis. Thus, acetyladenylate appears to play a role in generating clockwise rotation at the level of CheY or the motor.
Acetate-CoA Ligase↗