Motile Behavior
of Bacteria
E. coli,
a self-replicating object only a thousandth of a millimeter in size,
can swim 35 diameters a second, taste simple chemicals in its environment,
and decide whether life is getting better or worse.
-- Howard
C. Berg
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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. coli’s standard accessories
include particle counters, rate meters, and gear boxes. This microorganism
is a nanotechnologist’s dream. I will discuss the features that make it
so, from the perspectives of several scientific disciplines: anatomy,
genetics, chemistry, and physics.
What made work on
the behavior of E. coli possible? The tale has two geneses. One involves
light microscopy and begins in the 17th century, when Antony van Leeuwenhoek
first observed swimming bacteria.1 (See box 1.) The other involves
molecular genetics and begins in the 20th century, when Joshua Lederberg
demonstrated that bacteria have sex, as evidenced by their genetic recombination.2
(See box 2.) Lederberg studied E. coli and Salmonella typhimurium,
two closely related organisms. They are the principal subjects of work
now being done on bacterial chemotaxis (the motion of bacteria toward
chemical attractants or away from chemical repellents). That work has
yielded an important model for understanding the behavior of cells at
the molecular level.
Anatomy
of E. coli
E. coli (like S. typhimurium) is a cylindrical organism
with hemispherical endcaps (as figure 1 shows). The cell, which weighs
only 1 picogram, is about 70% water. Some strains are flagellated and
motile; others are nonflagellated and nonmotile. When motile cells are
grown in a rich medium (such as salts plus a mixture of amino acids),
they swim in the direction of their long axis at a rate of about 35 diameters
per second, often changing course but rarely stopping. The chromosome
of E. coli consists of a single double-stranded chain of DNA about
700 times longer than the body of the cell. There are 4,639,221 base pairs
specifying 4,288 genes, most of which encode proteins.3 The
functions of only about 60% of these proteins are known. About 50 different
kinds of proteins are required to produce the cell’s chemotaxis, roughly
half for the assembly of flagella and half for behavioral control. When
E. coli grows, it first gets longer and then divides in the middle.
In a sense it is immortal, because the mother cell is replaced by two
daughters, essentially identical to the daughters of the previous generation.
The molecules of DNA in the members of a given set of descendants are
identical except for mutations, which occur spontaneously for a given
gene, at the rate of about 10-7 per generation.
If well fed and held
at the temperature of the human gut (37 °C), E. coli can synthesize
and replicate everything it needs to make a new copy of itself in about
20 minutes. Thus, if we start at noon today with one cell (and lots of
food), by noon tomorrow there will be 272 = 4.7x1021 cells—enough
to pack a cube 17 meters on a side! This replication rate explains why
single cells dispersed on the surface of hard nutrient agar soon become
mounds of cells (colonies) a millimeter or so in diameter and why, in
soft agar, the motile progeny of a single cell soon populate the entire
plate.
Genetic
analysis
A fully functional cell line, or strain, found in the wild is called a
wild type. If a mutant cell is found that is missing a particular function,
the gene carrying the mutation is named for that missing function. For
example, a che gene is one encoding a protein (polypeptide) required
for chemotaxis. A cell with such a defect makes flagella and swims,
but it does not respond normally to chemical stimuli. The first gene of
this type to be identified is called cheA (in italics), the second
is called cheB, and so on through the alphabet. When the protein
encoded by the gene is identified, it is called CheA (capitalized and
in roman type).
In bacterial chemotaxis,
besides the che genes, we encounter fla genes, so named
for their defects in the synthesis of flagella (these genes are
now called flg, flh, fli, or flj, because
there turned out to be more than 26). There are also mot genes,
named for defects in motility, or generation of torque. And there
are a variety of genes that specify specific chemoreceptors; one, for
example, tar, is a gene encoding the chemoreceptor Tar, which is
so named because it mediates taxis toward the amino acid aspartate
and away from certain repellents. The soft-agar plate shown in
box 2 was inoculated with wild-type cells at the top, cells of a tsr
(the s stands for serine) strain at the right, cells of
a tar strain at the bottom, and cells of a smooth-swimming che
strain at the left.
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Figure
1. Motile bacteria.
Left: Three-cell cluster of Salmonella
typhimurium. The cell bodies are about 1 mm
in diameter and 2 mm long, but they appear
fatter because of the electron-dense stain of phosphotungstic acid
that was used to prepare the cells for transmission electron microscopy.
The flagellar filaments have a wavelength of about 2.3 mm
but are distorted by interactions with the substrate. The filaments
are 23 nm thick. (Image by Chi Aizawa, Teikyo University.)
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Above:
Scale drawing of Escherichia coli, showing one flagellum,
truncated, and three porins (protein channels that allow the entry
of water-soluble nutrients). A typical cell has up to six flagella
and hundreds of porins. The cell body is 2 mm
long; the multilayered wall is about 30 nm thick.17 The
outer membrane is made of polysaccharides and lipids, with the sugar
chains pointing outward. The inner membrane’s phospholipid bilayer
core resembles the membranes that enclose human cells. This membrane
is traversed by proteins involved in sensory transduction and in transporting
materials and harvesting energy. It constitutes the main permeability
barrier that enables the cell to retain the chemicals that make up
the cytoplasm—DNA, RNA, proteins, and various water-soluble molecules
of lower molecular weight.
Between the
inner and outer membranes is a porous, gauzelike layer of peptidoglycan
(polysaccharide chains cross-linked by peptides), which gives the
cell its rigidity and cylindrical shape. When the assembly of this
polymer is blocked by an antibiotic such as penicillin, a growing
cell cannot cope with the high osmotic pressure of its cytoplasm,
and it blows up. The intermembrane space, the periplasm, contains
a variety of proteins that either bind molecules that interest the
cell (such as sugars) or destroy molecules that pose a threat (such
as foreign DNA).
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The
flagellum
The flagellum is an organelle that has three parts (as figure 2 shows).
There is a basal body consisting of a reversible rotary motor embedded
in the cell wall, beginning within the cytoplasm and ending at the outer
membrane. There is a short proximal hook, which is a flexible coupling
or universal joint. And there is a long helical filament, which is a propeller.4
Torque is generated between a stator connected to the rigid framework
of the cell wall (to the peptidoglycan) and a rotor connected to the flagellar
filament. The proteins MotA and MotB are thought to constitute the elements
of the stator; FliF, G, M, and N (the MS and C rings) those of the rotor;
FlgB, C, F, and G those of the drive shaft; and FlgH and I (the L and
P rings) those of the bushing that guides the driveshaft out through the
outer layers of the cell wall.
Figure
2. Bacterial
motor and drive train.
Above: Rotationally averaged reconstruction
of electron micrographs of purified hook-basal bodies. The rings
seen in the image and labeled in the schematic diagram (right)
are the L ring, P ring, MS ring, and C ring. (Digital print
courtesy of David DeRosier, Brandeis University.)
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The proteins that
make up the flagellum are present in multiple copies. For example, there
are about 5000 molecules of FliC (also called flagellin) per helical turn
of the filament, which can have as many as six turns. The MS, P, and L
rings each contain about 26 copies of FliF, FlgI, and FlgH, respectively.
There appear to be eight stator elements (complexes of MotA and MotB),
each of which exerts a similar force.
If one fixes a wild-type
cell to a glass slide by one of its flagellar filaments, the motor at
the base of that filament spins the cell body at about 10 Hz. This technique,
known as tethering, was developed by Mike Silverman and Mel Simon at the
University of California, San Diego. If one tethers a paralyzed cell,
such as one with defective MotB, the cell body simply executes rotational
Brownian movement, like a mirror on a galvanometer fiber. However, if
wild-type MotB is made—for example, if a copy of a wild-type gene is added
to the cell and expressed—then rotation resumes. The good MotB proteins
that are made replace the bad ones, and the cell speeds up. Changes in
speed are abrupt, generating a speed–time plot in the form of a staircase
with eight steps of equal height. The flagellum is assembled from the
inside out, with the axial components exported through a central channel.
The filament grows at the distal end, with molecules of FliC added under
the distal cap, which is made of FliD. The growth process is subject to
exquisite genetic control. FliC, for example, is not made until the assembly
of the basal body is completed. When it is completed, the same apparatus
that exports FliC pumps an inhibitor of late-gene transcription out of
the cell. This removes the inhibition.
The motor is driven
by protons flowing from the outside to the inside of the cell (except
for marine bacteria and bacteria that live at high pH, where sodium ions
are used instead). The source of energy is a transmembrane electrical
potential or pH gradient (or both), generated by respiration for cells
grown aerobically. MotA and MotB form a transmembrane channel. Proton
translocation is thought to cause the cytoplasmic part of MotA to move
or change its shape and exert a force on FliG, thereby driving the rotor.
In each cycle of this process, the rotor advances by one or more steps,
which are of equal angular increment. From an analysis of fluctuations
in the period of rotation (assuming exponentially distributed waiting
times between steps), the number of steps is found to be at least 50 per
revolution per stator element. As the number of stator elements increases
toward eight, the rotation becomes smoother. The number of steps for the
intact motor is at least 400 per revolution. Individual steps have yet
to be resolved: They are filtered out by the elastic tether.
Detecting
chemical gradients
The motor runs either clockwise (CW), as seen by an observer standing
on the outside of the cell looking down at the hook, or counterclockwise
(CCW), with protons continuing to flow from the outside to the inside
of the cell. Switching direction involves the proteins FliG, M, and N.
In a cell wild type
for chemotaxis, CW and CCW modes alternate (with exponentially distributed
waiting times). When the motors turn CW, the flagellar filaments work
independently, and the cell body moves erratically with little net displacement;
the cell is then said to “tumble.” When the motors turn CCW, the filaments
rotate in parallel in a bundle that pushes the cell body steadily forward,
and the cell is said to “run.” The two modes alternate. The cell runs
and tumbles, executing a three-dimensional random walk, as shown in figure
3.
When different flagellar
motors in the same cell are studied under conditions in which they cannot
interact mechanically, they change directions independently. Yet, when
a flagellar bundle drives the cell forward, all of the motors have to
rotate CCW. The events that bring about this coordination are not yet
understood. The mean run interval is about 1 s, whereas the mean tumble
interval is only about 0.1 s. Both of the times are exponentially distributed.
Box
1. Light microscopy
Antony van Leeuwenhoek, using a single-lens microscope,14
was intrigued by “animalcules” (little animals) that he saw in his
well water. He wanted to know whether they might survive exposure
to pepper, so he ground up some and added it to a sample. The number
of animalcules waxed and waned until 6 August 1676, when he made
a discovery:
" I
now saw very plainly that these were little eels, or worms, lying
all huddled up together and wriggling; just as if you saw, with
the naked eye, a whole tubful of very little eels and water, with
the eels a-squirming among one another: and the whole water seemed
to be alive with these multifarious animalcules. This was for
me, among all the marvels that I have discovered in nature, the
most marvellous of all; and I must say, for my part, that no more
pleasant sight has ever yet come before my eye than these many
thousands of living creatures, seen all alive in a little drop
of water, moving among one another, each several creature having
its own proper motion".1
He was looking
at a spirillum, probably Spirillum volutans, the large bacterium
shown in the accompanying sketch. Leeuwenhoek never saw its flagella.
Those organelles of locomotion were first seen on Chromatium
okenii, another large bacterium, by Christian Ehrenberg in 1836,
and later on S. volutans by Ferdinand Cohn in 1872. The subject
of bacterial behavior was taken up systematically in the 1880s by
the physiologist Theodor Engelmann in Utrecht, and by the botanist
Wilhelm Pfeffer in Tübingen. They studied the responses of various
species of bacteria to light, oxygen, salts, and a variety of nutrients.
Pfeffer, who thought that bacteria could steer toward or away from
a chemical source, coined the term “chemotaxis” to describe their
attraction or repulsion. The role that flagella play in that response
was examined in detail after dark-field condensers of high numerical
aperture were developed, beginning in 1909 with work done by Karl
Reichert and culminating in 1920 with the work of Paul Metzner,
who described the motion of flagellar bundles of S. volutans
in stunning detail.15
S.
volutans has two flagellar bundles (as shown in the sketch),
each composed of about 25 flagellar filaments. Here, the cell is
swimming from left to right. Its body is helical. The bundle on
the left is in the tail configuration; the one on the right is in
the head configuration. When the filaments change their directions
of rotation, the bundles switch their configurations and the cell
moves in the opposite direction. An Escherichia coli bacterium
is shown below the S. volutans for comparison. As many as
six flagellar filaments arise at random from the sides of the E.
coli cell and form a bundle that appears near one pole. Rotation
of the filaments in the bundle pushes the cell forward. When the
bundle changes its orientation, the cell goes off in a new direction.
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Although the change
in angle generated by a tumble is approximately random, there is a slight
forward bias. When, by chance, a cell moves up a spatial gradient of a
chemical attractant or down a spatial gradient of a chemical repellent,
runs are extended. When, by chance, it moves the other way, runs revert
to the length observed in the absence of a gradient. Thus, the bias in
the random walk that enables cells to move up or down gradients is positive.
Finally, the behavioral
response is temporal, not spatial. E. coli does not determine whether
there is more attractant, say, in front than behind; rather, it determines
whether the concentration increases when it moves in a particular direction.
Studies of impulsive stimuli indicate that a cell compares the concentration
observed over the past 1 s with the concentration observed over the previous
3 s and responds to the difference.5
Constraints
imposed by physics
Look again at the runs in figure 3. They are not quite straight. The cell
is subject to rotational Brownian movement that causes it to wander off
course by about 30° in 1 s. After about 10 s, it drifts off course by
more than 90° and “forgets” the direction in which it was going. This
makes earlier measurements irrelevant and sets an upper limit on the time
that the cell has to decide whether life is getting better or worse. A
lower limit is set by the time required for the cell to count enough molecules
of attractant or repellent to determine their concentration with adequate
precision. The number of receptors required for this task proves to be
remarkably small, because diffusion of the molecules to be sensed enables
them to be sampled by different points on the surface of the cell with
great efficiency.6
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Figure
3. Movement.
This stereo plot shows about 30 s in the life of one Escherichia
coli K-12 bacterium swimming in an isotropic homogenous medium.18
The track spans about 0.1 mm, left to right. The plot shows 26 runs
and tumbles, the longest run (nearly vertical) lasting 3.6 s. The
mean speed is about 21 mm/s. To see this
plot in three dimensions, look at the left image with your left eye
and the right image with your right eye, and relax your eye muscles
so that the two images overlap. A stereoscope (pair of lenses) helps. |
Flagellar mechanics
is dominated by viscosity, not inertia: The Reynolds number is low,7
less than 10-4. So flagella generate thrust by using viscous
drag. The viscous drag on a thin rod is about twice as great when the
rod moves sideways as when it moves lengthwise. As a result, when oriented
slantwise and pulled downward through a viscous medium, a rod moves to
one side. (Geoffrey Taylor of the University of Cambridge in the UK showed
this in his 1967 demonstration lecture by dropping thin cylinders in syrup.8)
So, when a cell rotates a helical filament (in effect, a series of slantwise
rods), it has to push as well as twist. In reaction the cell body translates
and rolls. This scheme may not be very efficient, but it works!9
Processing
chemical signals
The molecular machinery by which E. coli modulates the direction of rotation
of its flagella is complex, as indicated in figure 4, which shows only
the aspartate receptor. (For general reviews of the mechanism, see references
10 and 11.) Three stages of the change-of-direction process can be distinguished:
- Chemoreception:
Aspartate diffuses to Tar and binds.
- Signaling: The
aspartate binding reduces the rate at which CheA phosphorylates CheY.
Because CheY-P is continuously being dephosphorylated by CheZ, the concentration
of CheY-P falls and less CheY-P binds to the base of the motor. When
CheY-P binds to the motor, the CW state is stabilized and the CCW state
is destabilized; with less CheY-P, the probability that the motor will
spin CCW increases, and runs are extended.
- Adaptation: The
decreased catalytic activity of CheA renders CheA less susceptible to
the action of CheB-P and also reduces the concentration of CheB-P, which
dephosphorylates spontaneously. Methyl groups are added to Tar by CheR
and removed by CheB-P. Hence the methylation level goes up. This tends
to restore the catalytic activity of CheA.
Figure
4. Chemotaxis machinery.
This diagram shows some of the components required for chemotaxis
toward the amino acid aspartate. Information flows from the outside
of the cell (shown at the top) by way of porins, the periplasmic space,
and the cytoplasmic membrane, to the inside of the cell (shown at
the bottom), and then to the flagellar motors (not shown). Dashed
arrows indicate physical displacement of chemicals by diffusion. Solid
arrows indicate chemical modifications of proteins—phosphorylation
or methylation. The cytoplasmic components, all Che proteins (CheW,
CheA, CheR, CheB, CheY, CheZ), are identified by their fourth letter
only. The receptor complex consists of two molecules of Tar, two of
W, and two of A, with Tar spanning the cytoplasmic membrane. Chemoreception
is depicted in orange, signaling in green (for “go”), adaptation in
red (for “stop”). Tar is a protein required for taxis toward aspartate
and away from certain repellents. ATP is adenosine triphosphate, the
phosphate donor. SAM is S-adenosylmethionine, the methyl donor. The
other chemicals shown are ADP, adenosine diphosphate; SAH, S-adenosylhomocysteine;
CH3, the methyl group; CH3OH, methanol; and
P, inorganic phosphate. |
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When a cell is exposed
to a step increment in the concentration of aspartate, it adapts completely:
Eventually the CheY-P concentration returns to its initial value and the
cell goes back to swimming just as it did before. However, in a cell exposed
to a ramp in the concentration of aspartate, adaptation will fail to catch
up and the cell will continue to swim more smoothly. In effect, a cell
makes temporal analyses by comparing the occupancy of the aspartate binding
site to the level of methylation of the receptor’s cytoplasmic domain.
The occupancy of the binding site reflects current conditions, whereas
the level of methylation reflects past conditions. The cell is able to
respond to changes in the ambient concentration by comparing these two
indicators. Adaptation is a universal feature in sensory physiology; for
example, you are not aware of the pressure exerted by your chair on the
seat of your pants unless someone calls it to your attention or unless
it changes suddenly.
The sensory network
shown in figure 4, complex though it is, is relatively well understood,
and theorists are beginning to model it. An interesting recent development
is the realization that the way in which the network is interconnected
may determine which features are robust—that is, which features are relatively
insensitive to variations in the concentrations of the proteins that compose
the network. In an organism as small as E. coli, such variations
are inevitable. For example, when a cell divides, soluble proteins are
partitioned at random. Because most of the proteins involved in signaling
and adaptation are soluble and are present in only a few hundred copies
per cell, their concentrations vary. Adaptation back to a particular mean
run interval appears to be such a robust feature.12
X-ray studies have
revealed structures of several of the proteins (or domains of the proteins)
involved in chemotaxis, including one of the components of the flagellar
motor.13 Given such structures, we should be able to work out
how the proteins function.
Box
2. Molecular genetics
Joshua
Lederberg chose Escherichia coli strain K-12 for his experiments
on genetic recombination. He did the work at Columbia University
and Yale University and published his findings in 1946.2
Later, at the University of Wisconsin, he manipulated genes for
flagella, not of E. coli but of a closely related species,
Salmonella typhimurium, which is encountered in food poisoning.
The flagella of Salmonella were known to be efficient antigens
that could be used to distinguish among pathogenic strains. When
genes for flagella were moved into a nonmotile strain, motile recombinants
could be readily isolated, because they swarmed through soft agar
(as shown at the top of the figure) while the nonmotile variants
remained at the point of origin of the inoculation (as shown at
the left in the figure). (The use of agar in bacteriology dates
from 1882, the swarm technique from 1897.)
In the 1960s,
Tetsuo Iino, who had studied with Lederberg and then moved to the
National Institute of Genetics in Mishima, Japan, collaborated with
Sho Asakura and Goro Eguchi of Nagoya University to publish a series
of elegant papers on the assembly of flagellar filaments. The researchers
found that the structures could be sheared off of bacteria, dissolved
(by heating, for example), and then recrystallized in a test tube.
The assembly process could be analyzed by observing the seeds of
one antigenic type (or polymorphic form) interacting with monomers
of another. At about the same time, Julius Adler, a biochemist at
the University of Wisconsin, chose E. coli strain K-12 to
use in studies of bacterial chemotaxis. Although he was fascinated
by the behavior of butterflies, he sought a simpler system in which
to learn about molecular mechanisms. In 1969, he published a now-classic
paper in which he showed that bacterial chemotaxis is a matter of
esthetics, not material gain.16 Normal cells are attracted
by nutrient analogs that they cannot metabolize, and mutant cells
that have lost the ability to take up or metabolize nutrients still
find them attractive. While it is clear that taste for a particular
chemical evolved because that chemical was useful to the cells,
the behavioral decision is made on the basis of taste alone.
The accompanying
figure shows a soft-agar plate 8.5 cm in diameter containing a mixture
of 20 amino acids. By stabbing the agar with a sterile toothpick
dipped in a cell suspension, four E. coli strains were inoculated
in this plate. The cells were allowed to grow for about 8 h at 30
°C. Then the plate was illuminated slantwise from below and viewed
against a dark background. Normal cells (top) first consume serine,
generating a spatial gradient of serine that they chase across the
plate, forming the outer ring. Then the cells left behind consume
aspartate, forming a spatial gradient of aspartate that they chase
across the plate, forming the inner ring. Cells of the strain shown
at the right have lost their ability to taste the amino acid serine,
and so the outer ring is missing. Cells of the strain shown at the
bottom have lost their ability to taste the amino acid aspartate,
and so the inner ring is missing. Cells of the strain shown at the
left can swim but are unable to back up; they become trapped in
blind alleys in the agar. Adler perfected the use of this technique
for identifying behavioral mutants. (Photo courtesy of Sandy
Parkinson, University of Utah.)
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Motor
mechanics
I am particularly interested in motor mechanics. What makes the motor
go? How much torque can it develop at different speeds? How does it change
its direction of rotation? What are the mechanical interactions between
flagellar filaments that coordinate different motors on the same cell?
Here is a sample experiment. Because shearing a dense cell suspension
causes the filaments to tangle and fracture, we can break off most of
a cell’s flagellar filaments and then cement the cell body to a glass
slide. If the cell makes filaments that tend to stick to everything (because
of a particular mutation in fliC), then it is easy to attach a
latex bead to one of the flagellar stubs. If the bead is relatively small—say,
400 nm in diameter—it will spin relatively rapidly, because of its small
frictional drag coefficient. We can measure its rotational speed with
a weak optical trap by means of back-focal-plane interferometry. The torque
required to rotate the bead at angular velocity W is just bhW, where b
is a geometrical factor and h is the viscosity of the medium. The viscosity
can be changed. Then, by plotting hW versus W, we can obtain a measure
of relative torque versus speed.
At room temperature,
the torque–speed curve for the motor of E. coli is relatively flat,
falling about 10% between stall and 200 Hz. Thereafter it declines linearly,
reaching zero torque at about 300 Hz. In the high-torque, low-speed regime,
the torque is insensitive to changes in temperature or hydrogen isotope;
evidently rates of diffusion of internal components or of proton association–dissociation
reactions do not matter. In the low-torque, high-speed regime, on the
other hand, the torque falls substantially as the temperature is lowered
or as H2O is displaced by D2O. Now, rates of proton
association–dissociation reactions do matter. What is surprising is that
the region of transition between the two regimes is so abrupt.
Other
worlds
But I have spoken only of flagellated organisms, of which E. coli is the
pre-eminent model. There are other bacteria that move without flagella,
either through liquids or at liquid–air or liquid–solid interfaces. Their
means of locomotion are poorly understood.
I thank John Menninger
for comments on the manuscript. Work in my laboratory has been supported
by the National Institutes of Health, the National Science Foundation,
and the Rowland Institute for Science.
Howard
Berg is a professor of molecular and cellular biology,
and of physics, at Harvard University in Cambridge, Massachusetts,
and a member of the Rowland Institute for Science. |
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©
1999 American Institute of Physics
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