( applause ) >> Thank you.
So, the title has changed a little bit.
I sent in a new title, but I noticed that it didn't make it
in time to get posted. I apologize if you were looking
forward to something that was a little bit more tame.
But what I want to talk to you about tonight is kind of an
emerging area, and one that we're very interested in.
I think it's a very fascinating story, and something that
I find quite exciting. And I hope to convey
that enthusiasm to you. So the title was a little racy.
This is not exactly what I'm going to talk about, which is,
you know, curves in this sense. But really, what I'm going to
tell you about is something that I find very sexy,
which is E. coli. This shows a very
high-resolution image of a cell of bacteria.
We're very interested in studying and understanding
its behavior. There's a really long tradition,
a rich history of work on bacterial behavior, of course,
at Madison, through the work of Julius Adler and other people.
So before I start, I should tell you about--
I still do experiments. Usually, we'd say, I'm going to
tell you about the people that did the experiments.
In this case, I actually did many of the experiments.
But these are the students in the lab.
And these are some of the people that have so generously
given us money so far. So I'll start with that.
This is essentially what I'm going to tell you about.
There are four parts to it. Four Acts.
Act I, is I'm going to tell you a little about bacterial cell
shape and why you should care, and why I care.
Then I'm going to tell you about a technique that we've worked on
that allows us to control bacterial cell shape.
Then I'll tell you about why we want to do that.
I'll show you that it's a tool that allows us to look at
how shape controls the movement of bacteria.
That's something that's called low Reynolds number.
Tonight, you'll learn some biology, chemistry and physics.
And maybe some fluid mechanics. Then in the fourth part,
I'm going to tell you about how we use this technique to start
thinking about how shape has evolved, how and why
bacteria have specific shapes. So this is Act I.
This is a very famous movie in biology.
What you're watching is a movie from David Rogers.
He essentially drew blood from his finger, mounted it
on a glass slide, put it under the microscope.
And you're watching a neutrophil.
This is a cell in your immune system.
It's chasing down a single bacterium.
This bacterium is in the genus Staphylococcus.
This happens to be a human pathogen.
These globs here are red blood cells.
The movie is looped. The point here is to show you
that a lot of eukaryotic cells are what we call pleomorphic.
They don't have a specific shape.
It's very dynamic. They're constantly changing
their shape. That's what you see here.
And in fact, it's really interesting because this cell--
It's got it now. And what you see,
it's actually hard for-- The fluids behave in weird ways
when you're this small. Look, it's actually pushing
the fluid. That's pushing the little
bacterium, okay? So eventually, the bacterium
sticks on the surface, and the neutrophil pops on top of it.
But that's one thing that we're going to talk about tonight.
Fluids behave in strange ways when you're this small.
In contrast, bacteria, largely, have very static shapes.
They're only a specific shape. They don't change
back and forth, typically. There are some oddballs that
we're very interested in, but those are really the minority.
These are the majority. These are a representation
of the different kinds of shapes you can expect to see
in the bacterial kingdom. So these are really
the three main shapes. You have spears.
You have rod-shaped cells. And there are these cells
that are spirillum. They're like your
telephone cord. These cells, just to give you
a calibration, one of these cells is about a micron.
So what does that mean? Your hair is about 100 microns.
You could fit 100 of these cells across your hair.
Your finger is about a centimeter.
You could put 10,000 of these cells across your finger.
They're quite small. The most important thing
on this slide is that shape is conserved.
It doesn't change. That means that a mother cell
starts off in time and produces daughter cells.
It does it very quickly. Every 30 minutes it produces
a daughter. So we inoculate.
We put a single motor cell in some liquid, culture it
over night. You come in the next morning
and there are billions of cells. They all have the same shape
as the mother. The shape is genetically encoded
at the level of the chromosome. We're really interested
in asking why and how has shape evolved.
Why do bacteria have these specific shapes you can kind of
see in the background here on the last slide.
We want to understand volume control.
Why do we get variations, skinny bacteria and huge bacteria?
We also want to understand, from a molecular level, how does
the cell control it's shape. What are the proteins
that are involved? What's the chemistry
that's involved in doing this? At this point, I think you
should be asking yourself, you know, two big words.
Who cares? Why is it so interesting
to understand the shape? Well, it turns out that from
what we know now, from the story that's emerging, many of the
players, the proteins that are involved in controlling
bacterial shape are essential for the cell.
That is, you get rid of those proteins and the cell dies.
So what that suggests is that as the story emerges, we'll find
new targets for the development of antibiotics or new mechanisms
for developing anti-microbial compounds.
This slide is a little busy. But the idea is to show you that
there's a lot of sophistication in bacterium.
Bacteria, 50 years ago, were thought by many people,
many biologists, just to be a membrane, essentially,
a cell wall that surrounded a sac of cytoplasm.
That's the thick, goopy stuff that has lots of proteins,
in which there was a chromosome, and that was it.
There wasn't much more sophistication than that.
That turns out to be wrong. This shows you four examples
of how that's wrong. It turns out that in bacteria,
there's a lot of organization. Things go to specific places.
They're not just random. Proteins are not just randomly
floating around. They're put in specific places
in the cell to do specific things.
It makes sense. So this shows--
It's really difficult to see, but it's an analog of a class
of eukaryotic proteins that we call actin.
It forms protein filaments. That is, the proteins aggregate
and polymerize into a filament. The filament, we know plays
a role in controlling the rod shape of some species
of bacteria. But we don't know how it works.
This is another example of an actin homolog
that forms a polymer. So it polymerizes.
And what it does is it grabs two different pieces of DNA.
It makes sure that a mother cell, as it's dividing,
gets one copy to the mother cell and one copy
to the daughter cell. This piece of DNA is required
for the cell to survive. It conveys antibiotic
resistance. Imagine you're a mother cell.
You have a few pieces of this plasma DNA.
You want to give a copy to your daughter cell.
How are you going to do it? Well, you don't have fingers,
right? If you just rely on random
diffusion, the daughter cell may end up with every copy.
And then the mother cell is done.
So this system actually is a way of pushing copies along
the long axis of the cell so that one gets over here,
one gets over here, and the cell divides
right down the middle. It's a mechanism for moving
things in cells and bacteria. This shows a protein that's
an analog or a homolog of a class of proteins that we call
tubulins and eukaryotes. Bacteria forms a ring right
at the center where the cell is going to divide after it's
made the daughter cell. It's got to pinch off.
And what this does, it defines the center of the cell,
and recruits a lot of proteins that then cause the cell
to essentially divide. And finally, there's a homolog
of intermediate filaments. And this is involved
in controlling curvature. So the pink protein here is
a filament that forms along the inner ridge of the cell
and causes the cell, in this case the genus is Colibacter,
to buckle. The point here is not to give
you an entire 50-minute talk on why these curvings are
important, but to tell you that here's a class of proteins
that are very sophisticated. They form very sophisticated
structures. They're found in bacteria.
They're very dynamic. And these same proteins are the
components in eukaryotic cells that are responsible for
making them so pleomorphic, like in that movie we saw
of the neutrophil. Even though these are there
and they're dynamic, they're not involved in controlling
a dynamic shape because bacteria don't have that.
They have a static shape. So cell shape must come
from something else as well in bacteria.
And it does. It evolves the cell wall.
The cell wall of a bacterium is very unique.
This shows one kind of bacteria that we refer to
as gram negative. We can think of many different
genera of bacteria that fall into this class.
It's like a capsule. It's got that kind of
a vitamin shape. If you take a cross section
of the cell wall and you pull it out,
it looks a little bit like this finger sandwich.
The finger sandwich has two yellow layers and this
pink layer that look a little bit like --
And if you look at this in detail and you blow it up like
a cartoon, this is what you see. The yellow layer here
is a lipid-bilayer. It's the same thing you find
in a eukaryote. It's like a membrane.
It is a membrane. And on the other side, you find
the same thing. You have another lipid-bilayer.
These two lipid-bilayers exist. They've sandwiched
this pink layer. The pink layer is actually
a polymer. It's a polymer of sugar.
It's what we refer to as the peptidoglycan.
The peptidoglycan is very important material for bacteria.
And it's a very fantastically-- It has fantastic
mechanical properties. Let me just leave it at that.
The peptidoglycan consists of these repeating rods.
I'm a guy, so I'm really bad with colors, so I hope you'll
excuse me. Maybe there's some guys out
there that are good with colors. But there's this salmon-like
color, maybe a peach color. Then there's this salmon color.
Peach and salmon. And the peach corresponds
to this monosaccharide called N-Acetyl Muramic acid.
The salmon color corresponds to another monosaccharide,
N-Acetyl glucuronic acid. These are linked together
inside of the cell and flipped into this space, and
then incorporated into this rod. So these disaccharides are lined
up and polymerized together using a series of enzymes.
The cell essentially makes a series of rods,
like these kids' toys. I like to play with kids' toys.
I like to use these to think about projects.
But it makes a series of rods. A series of rods, as you know,
is not a very mechanically stable structure.
If you apply pressure, right, they just deform.
So what you'd like to do is take these and cross link them
into something that looks like a mesh.
It turns out the bacteria have done that.
They then take these amino acids, these short peptides
on the side chain of this NAM residue.
They link these things together. So what they're essentially
making is a mesh. This is just kind of a rough
structure that was done by a very high resolution imaging
technique called nuclear magnetic resonance imaging,
or NMR. That was used to image fragments
that were made synthetically. And this doesn't represent
exactly what's in a cell, but it gives you an idea of the kind of
structure where you see this kind of honeycomb-like structure
in the peptidoglycan model. So that's what we assume this
to be. And this pore size
is about four nanometers. That means that things that
are four nanometers or smaller, actively can go through
this peptidoglycan. Things that are larger
probably can not. It turns out that we're finding
in the lab that larger things can go through, which we're
still not sure about. The peptidoglycan is probably
the most important material for the bacterium.
It's a cross linked sugar. It's a polysaccharide,
as I told you. It's essentially one--
You can think of this as one single molecule.
This is a gigantic molecule with a molecular weight of about
100,000 kD, which is very large. And in a bacterial cell wall,
in a gram negative bacteria, it's thick.
This is about 15 nanometers thick.
This is referred to as rigid, because this is the material
that conveys all the mechanical properties of the bacterium.
That is, we put a stress on the cell, the stress is essentially
dissipated at the level of the peptidoglycan.
It's mechanically stabilizing the cell.
So people have done very crude measurements of properties
of this material. The idea here is they use
what's called a scanning probe. It's essentially a record
player. I almost forgot what that was!
That's a joke. Actually, my kids have a record
player. So essentially, you have a
record player with a needle. Instead of having a needle that
you can see, this needle is so small you cannot see it.
It's several nanometers thick at the end.
It's tapered to a very, very thin, thin structure.
And what you can do with this, what they've done is they've
essentially made a groove in a slide.
They took pieces of the cell wall out of the cell and put it
on top of this groove. They then brought this little
tip in and pushed material down and released it.
They measured what happened to the material as they applied a
force to it and it was released. What they found was that there
was no hysteresis, which means that, you know, if I take
a sheet of aluminum foil, and if I was smart,
I would've brought one so I could show you--
You take a sheet of aluminum foil and I puncture it, right,
it forms some kind of structure. It's deformed.
Does it go back to its original structure?
No. And that's called hysteresis.
There's no return to the original structure.
Here, though, it's like you're pressing it down and it springs
right back up to its original position.
So we can't really think about it as rigid.
Because if it was rigid, it wouldn't have this property.
It's not like aluminum foil. If you measure what's called
the Young's modulus-- The Young's modulus is a slope
of a plot that's the stress versus the strain.
The stress is the amount of force you apply per unit area.
The strain is the amount of deformation that's caused
in the material. You get some number.
And you're probably thinking, oh, yeah, that's about right.
Ten to the seven newtons per meter squared.
I mean, I look at that, and it's just another number to me.
But what that correlates to, if you look at polymers--
And I'm cheating, because I don't have latex gloves because
I have an allergy to them now since I've worn them
for ten years in the lab. But it's a lot like
a piece of latex, okay. It's got this kind of property.
So it really is a flexible material.
Flexible, yet it protects the cell.
The question is, how does the cell control its shape with a
material that is semi-flexible. And there are two hypotheses
that we look at. One is that the peptidoglycan
is just a shell. It's like a blimp.
We call this the Goodyear Blimp model.
It's just a shell around the bacterium that has these
kind of cross linkers that support, or struts.
They support and keep the structure in a cylindrical
shape. The other possibility is that
the peptidoglycan layer is made, when the cell is making it,
it controls the way it's made, in that it controls the shape.
So the shape is encoded in the way the peptidoglycan
is synthesized and cross linked. So we think about
these two mechanisms. Now, to think about these
mechanisms, we need to have a technique for controlling
the shape of cells. We want to be able to engineer
and reshape the bacteria. This is the way
we came up with it. The idea is we take
a material-- Oh, and let me ask,
I've given talks to kids before. Am I okay?
Am I doing okay? Okay.
( inaudible ) Okay.
So the idea here is we take a material and we create a little
compartment for the cell. We call it a microchamber.
Microchamber, because it's got micron scale dimension,
so micro. It's a chamber, because it's
a little place for the cell to reside.
I'll tell you how we do that in a second.
But this just shows one of these.
We essentially print this into a polymer.
And the polymer is one that allows the cell to grow
and replicate. And then we put an individual
bacterium in the structure. Then we trick the cell.
We give it everything it needs to grow, but we essentially
tell it that it can never divide into a daughter cell.
So what this cell does, is it just grows longer and longer.
It grows into what we call a filament.
The issue is that it's confined.
It's in a compartment that's got a shape.
In fact, we control the polymer so that it's stiffer
than the cell wall. So something's got to give.
Does the cell give or does the material give?
If the material is stiffer, the cell has got to give.
That means the cell grows into the shape.
When we release it, it turns out that it retains that shape.
This is our trick for controlling a cell shape.
So we make these things. We put the cells in.
We filament the cells. We can do it using an antibiotic
like this one, or we can use genetics.
Then we take the cell out. We essentially take the ceiling.
There's a ceiling on this that we put on that I didn't
tell you about. We just pop the cell out
and it goes on its merry way. The reason that this works
is because when a cell grows, when a bacterium grows,
it doesn't just grow at one end. Imagine you're a mother cell
and you're going to grow. It doesn't just start growing
at one end and continue growing and divide.
It grows along the length of the cell.
That is, new peptidoglycan and cell wall is inserted along
the entire length of the cell. So what happens is in the
presence of this molecule that tricks the cell
so that it can never divide, the cell gets longer.
We started with one chromosome. Bacteria always have
one chromosome. It replicates,
now it's even longer. It's got two chromosomes.
Now it's even longer, it's got three.
And in fact, we can do this, and grow bacteria that are
millimeter length. This shows that the cell indeed
is inserting new cell wall along the length of the cell.
What this shows you is a bacteria--
This was the original bacteria. This is the daughter,
another daughter and another daughter that are enjoined.
The black spots are old peptidoglycan
that's been labeled. The white is new peptidoglycan.
So you can see that there's a lot of old peptidoglycan
at the ends. There's a reason for that.
That's a whole other talk. But essentially, new
peptidoglycan has been inserted along the length of the cell.
This region right here is a whole other talk, also.
That's really the take home point there.
So this is how we make these little components.
You might be asking, how do you make these little homes
for the bacteria. We use a technique
that was developed for making semiconductor chips
for computers. Essentially, what we do
is we take a wafer and we put a very thin layer of a polymer
on the surface. We can control the height of
that polymer using essentially a record player that we can
control the radio velocity of. Then we decide on what
our design is going to be. We draw it on a computer and we
print it onto a transparency, the same kind of transparency
that we've used for teaching, or we used a while ago
for teaching. And we put that on top of this
polymer, and you press it into very close contact.
Then you shine a light through. The light is only going to go
through the region that's transparent, right?
Here we've got these channels. This is what we want to make.
As the light goes through this region, it does chemistry
on the polymer. In fact, it cross links it
and makes it insoluble. The regions behind the black
part don't get exposed to light. So then after done, we dip it
in a solvent, in a liquid. The solvent dissolves away
all of this part and leaves behind this part,
which is now insoluble. We end up with the bottom half
of the structure, which is a silicon wafer, or a flat
surface with the structures sticking up from the surface.
Does that make sense? Now we take a polymer, such as
agarose, which is harvested from seaweed.
We put nutrients in it and everything that the cell
needs to survive. We cast it on top.
We essentially just pour the hot stuff on top
and then we cool it. Then we peel it away.
Now what we have is a layer of agarose that has these channels
embossed in the surface. Now we flip this over, and
we put cells on top of it. We also put agarose on a piece
of glass, just to support it. Then we smash,
or gently lay, excuse me-- We treat these bacteria
very ethically. We lay a cover slip
on top of that. The cover slip squeezes out
the excess liquid and traps the bacteria.
Then we put this on a microscope and we look at it.
What I'm going to show you are are not bacteria in channels,
they're bacteria in chambers. So you're looking down
on one of these structures. You can kind of see
the structure in the background. It's white.
These black things, these are cells.
These are individual bacteria that are trapped in these
compartments. And it turns out that we're
working with a strain of bacteria that are capable
of swimming, and actually swimming quite fast.
So what's important here is that the material allows
the cells to essentially breath. They've got to exchange gas.
They need nutrients. Nutrients have to diffuse
into the cell so the cell can survive.
Waste has got to diffuse out. This polymer is chosen
to allow that to happen. So the bacteria are actually
quite happy, as far as we know. These aren't just
empty channels. These fill up with water.
So to the bacteria, it's kind of like it's swimming.
It's like a little race track. So we let these cells grow
in these compartments. And what we find, we can do some
calculations and measurements. And we find out they grow
at the same rate they would grow in a bulk fluid.
So to the cell, if we do these kind of checks
to see if they're happy. We see the right phenotypes,
or the right types of behaviors that we would expect to see,
we do see them. This is a movie that shows you
a cell growing. It's a little hard to see,
because you've got to remember, this cell is about a micron,
so it's very pixilated. But these are 30-second
time points taken over 12 hours. You can kind of see it.
You see this bacteria is elongating.
And you'll see it. So you see it'll just keep
growing around and around until it fills that space.
That was the night I did not put my kids to sleep.
So using this technique, we can force bacteria into shapes
that they don't typically have. We can grow zigzag structures.
And I won't tell you why these are different, but
bacteria are smart, essentially. They find a way to kind of
grow out of the shape as best they can.
We can use sinusoidal shapes and circular shapes.
The important thing is that we've got to make the material
stiffer than the cell wall. As long as that's true,
it's going to happen. Now imagine we take cells
and we grow them in a circle. Imagine that we make the height
of this chamber tall enough that the cell can actually
grow around several times. Essentially what it is,
it's a coiled spring, right, that's what we have
at that point. Now we release them.
We take the top off and the cells: "Whoo, we're free!"
We wash them out. What we end up with
are elongated springs. So the structure
stays at the level of the cell. It doesn't just relax back.
So this is a technique for not only controlling the shape,
but for keeping the shape. The cell eventually will end up
retaining that shape. So you see lots of spring-like
bacteria that were made simply by growing these around
in this compartment, and then releasing them.
So we can impose a shape by physical constraint.
Essentially what we're doing is we're controlling, we believe,
how the peptidoglycan is inserted as the cell grows.
So the cells retain their shape as they grow in liquid.
Eventually, the cell will lose it's shape, and I'll show you
that in a second. The cells also divide.
So if we wash away-- If we turn on the machinery now
so that the cell can septate, the daughter cells start
breaking off and will swim away. So if we take 10,000 of these
shaped cells and leave them overnight, the next morning,
we come in, they're all small cells.
They actually-- What I haven't told you, is
they'll slowly lose their shape every time they replicate.
Let's imagine you're a bacterium that's got this shape.
Every time you replicate, you have to double in length.
I told you that every time you double in length,
new peptidoglycan is going to be inserted along the length
of you. So what happens is they slowly,
slowly relax over many, many replications.
The third part is something that's kind of a lot of physics,
but very interesting. The question is how does shape
affect motility. So this shows bacteria swimming.
This is real-time. This is not spread out.
You're watching fluorescently labeled bacteria swim.
I took this from Howard Berg. This is such a beautiful movie.
But what you see, is this is kind of the body
of the cell right here. And this long, curvy piece
is actually the flagella, that I'm going to tell you about.
Most of the cell, it looks like, is flagella.
The cells are very fast and they're capable of swimming.
Now this is how it works. This isn't a movie.
This is a cartoon. It's not mine.
But essentially there's a motor. There's a motor
in the cell wall. And if you look carefully, you
see these little white things flowing through it.
These are ions. So it's driven based on the flow
of protons into the cell. And there's a hook connected
to the motor. The hook is connected
to the flagellum. And a bacterium, E. coli, has
around four to five flagella, on average.
They're randomly distributed along the length of the cell.
These flagellum are thin polymer filaments.
They're about 20 nanometers wide.
They're about ten microns long. And the motor moves
very, very fast. So it moves at 100 Hz.
Does anyone know what something is that moves at 100 Hz?
( inaudible ) Electricity does.
And this is one example, a radial saw, a circular saw,
whatever you want to call it, operates at around 100 Hz.
You know that thing is flying, right?
It goes very, very fast. Let's just think about this
for a second and do kind of a calculation.
A motor like this probably pulls maybe ten amps, maybe 30 amps.
And an amp is a coulomb per second.
And a coulomb is about ten to the 18th charges.
So that means that a cell like this is running through
about ten to the 20th charged particles per second.
So in this case, it's electrons. Electrons are flying
through that motor to get the motor to turn.
Now the bacteria motor is not analogous at all.
But if you think about it in the same kind of light,
you can imagine that the number of charges that have to flow
through the motor to move it may also be very high.
And in fact, the bacteria spends an enormous amount
of energy controlling the membrane potential
so that protons can flow. Protons want to flow
through the motor, into the inside of the cell.
And in fact, I don't know. I can't remember what the number
is off the top of my head, but an enormous amount of metabolic
energy is spent by the cell just maintaining that potential
to move those motors. And these motors are not like
on and off. These babies are on for good.
So this thing is a huge energy drain for the cell,
but it allows the cell to move. Why would a cell want to move?
( inaudible ) To get somewhere.
Well, are they mating? Do bacteria have to mate?
No, they're self-replicating. That'd be awesome, right?
Make a million of yourself. ( inaudible )
To maybe get away from predators.
But also, really, to access food.
They want to find food. So the cells have to move faster
than the diffusion of things through liquids.
So they've evolved flagella to help them do that.
Okay, this shows what has actually happened
to the flagella. This is a cartoon again,
or a movie. And it's a 2-D representation
of a 3-dimensional image. That's why it looks kind of
just like the flagella are flapping back and forth.
They're actually rotating. So each one of these filaments,
the flagella, has a wavelength. Okay, it's like this.
And when all the motors are turning in the same direction,
the flagella bundle. And when the flagella bundle,
you can think of it as a propeller.
That's kind of what it looks like.
That's called a run. That's essentially the way
the cell moves itself forward. Now these cells don't have
rudders or steering devices. So you'd imagine they'd
need to be able to reorient their direction of movement.
The way that they do this is they turn at least one
of the motors the opposite way. So all of the motors are going
one way, and then one of the motors goes the opposite way,
or all the motors go the opposite way.
When that happens, it causes the flagella to splay apart.
And that's what we call a tumble.
It allows the cell to reorient. The reason it's reorienting,
remember, this is a micron. Can you imagine what it'd
be like to be a micron? You would feel water molecules
banging into you. You'd feel thermal energy.
It's thermal energy, this motion that causes the cell to get
turned around in this direction. And then it goes off again
on a run. And in fact, the person that
worked out this beautiful system is in biochemistry here,
Julius Adler. So there's something important
to tell you. Fluids are weird
if you're small. Fluids, you know, you pour them
in your hands. You wash your hands.
You swim in them. It's not a big deal.
It's quite different if you're very small.
And so, if you're a human and you swim, there's something
called a Reynolds number. Fluid dynamicists,
fluid mechanics use this to think about the forces
on an object that's moving in a liquid.
And essentially, the top here, which is the density of a liquid
row, "V" is the velocity, and "L" is the length,
and "U" us the viscosity of the liquid.
Essentially, the Reynolds number is dimensionless.
It's the ratio of the inertial forces to the viscous forces.
So if something is big, we say it's got a high Reynolds number.
So if a human is swimming, if you go through this calculation
and you say the average velocity of a human swimming is this,
and you know, density of water is one,
and viscosity is around this, and the length scale of a human
is around this number of meters. If you calculate
that Reynolds number, it's around ten to the 4th.
What that means that if that number is really big,
then it's the inertial forces that really matter.
So you're swimming along, you're doing the breast stroke,
and you stop. What happens to your body?
Does your body immediately stop? Let's say
you're an Olympic swimmer. Does your body stop?
No, what happens? ( inaudible )
It glides. And as it's gliding,
you're losing your momentum through this inertia.
Now for a bacteria, a cell of E. coli, we could do
the same calculation. You go just in the opposite
direction. You're ten to the minus 4th.
So it's a very low Reynolds number.
And in this case, viscosity is really the determining step.
So what this means is that a bacterium swimming along,
okay, it's flagella moving. Let's say it could stop
its flagella. What would happen to it?
The bacterium would stop so fast, it would lose it's inertia
immediately to the fluid, that it would only coast
less than one angstrom, which is like smaller
than the radius of an atom. It loses its inertia
immediately. So for a bacterium, it's not the
inertial forces that dominate, it's the viscous.
That turns out to be really important.
What if you wanted to do the experiment and see what
it felt like to be a bacterium? What you could do is you could
put yourself in a swimming pool, and you could fill it up
with molasses. And you would not be able
to move your arms any faster than one centimeter per minute.
That's what it would feel like if you wanted to be
at a very low Reynolds number. So let me ask you this.
If you were a bacterium, and I like to think of myself
as a bacterium, what shape would you want to be?
You're moving, right, you've got those flagella.
They're like a little propeller. They're turning.
What shape are you going to want to be?
I've got some choices for you. Do you want to be "A"?
Do you want to be "B"? It's actually
quite a lovely car. I'd like to be "B".
Do you want to be this Ferrari? How many people say "C"?
How many people say "B"? How many people say "A"?
I'm with you. We've got some of each.
And I had to put this in, because we're in this area
of the country. ( laughter )
What about "D"? ( inaudible )
We've got a "D" taker in the crowd.
I thought you'd be a "D" man. Okay, well, what if I told you
it probably doesn't matter? The reason is because it has to
do with the physics of fluid. There's this thing called
slender-body theorem, which is fluid mechanics theory,
which we're really not going to go into.
But the idea is if you do an approximation of this,
and you say, okay, I have a rod. This is my rod.
And it's got a radius, "A", so that's the radius of my rod.
It's got a length, "L". And I'm going to move it through
fluid this way at a velocity. Then the force approximates
to something like this. So, two-pi times the viscosity
times the length times the velocity,
divided by a natural log, blah, blah, blah.
So that's the first. What happens if you took it
and rotated it this way? And you pushed it through
the fluid this way? Well, it turns out that it
only scales by essentially a factor of two higher.
That means the force changes only by a factor of two
as you push it this way. Of course, it depends on the
length and stuff like that. But really, it's just changing
by a factor of two. That's a little bit surprising.
That really says that you could be a semi going this way,
or you could be a semi turning yourself on the side
and go this way, and the amount of force it takes
that you have to generate to move yourself through that fluid
at that velocity, really varies very little.
Surprise. So what does the force mean
in terms of velocity? Well, you can go through these
equations called Navier-Stokes equations,
which we're not going to do. You can do some approximations,
because based on, you can take the time components out.
What you end up with is that the velocity essentially
scales as the force. So you can approximate
the velocity as the force. What that means is that
the velocity going this way, at a constant force,
and the velocity going this way at a constant force
as you turn sideways, only changed by a factor of two.
So there's really not a big hit, if you've got a weird shape.
So it's not that different. So that means that shape
doesn't really matter. You could be a huge E. coli
traveling on your side, traveling on your back,
going the wrong way, and you could still move very fast.
But there's a catch, and that's Act IV.
It's kind of corny I did these Acts.
I usually say, "Part One, Part Two."
It's always the same, so I thought I'd try
something different. So this is a crazy idea
that we're thinking about. The question of how shape
has evolved and why bacteria have kind of a streamlined
looking shape. Isn't a cylinder
kind of streamlined? It looks kind of streamlined
to me. The question is
why do they have that. Here are some characteristics
of E. coli or bacteria that are motile.
First, remember that flagella take a lot of energy.
Those things are always on. Ten to the 15th charges
are probably passing through the motor per second.
That's a lot of energy. The cell has to bundle
the flagella. If the flagella don't bundle,
the cell doesn't go. And remember, the flagella are
kind of randomly distributed along the length of the cell.
It's not like there was a little landmark and the bacteria says,
"I'm going to put one there, and put one there..."
They're just more or less randomly distributed
along the side. So we have this hypothesis.
And that is that shape didn't really evolve
to make this so streamlined. It evolved to optimize
the bundling of the flagella. It's like if you had
eight pistons. You know, if all your pistons
are firing at strange time intervals and you can't control
it, your engine is going to-- Excuse my French.
It's going to suck, right? But if you can control
the way those are fired, then you can get your useful workout
of an engine. So let's test this hypothesis.
We have this way of controlling shape.
We can engineer curvature in bacteria.
So let's see if we can control cell shape and measure
the translational velocity of different shapes
and see what happens. So that's what we did.
So we took wild type cells. This is our control.
They're fast. They swim at about ten
to 20 microns per second. Okay, now we take a cell and
we engineer it into a spiral. These cells
are really not very happy. You can see, if you calculate
the translational component of velocity, it's zero.
These cells just kind of randomly tumble around.
The flagella are there, because we can image them and see
what they're doing over time. They're there
and they're turning, but the cell's not translating.
We can do the same thing. We've allowed these cells
to grow. If we'd taken one of these
springs and we let it grow over time, it elongates and
become something that looks-- I'm getting ahead of myself.
If we take a crescent and we watch it.
You can see it actually translates.
If we do this for 50 cells, and we calculate an average
translational velocity, it's about three microns per second.
So we're getting, kind of, maybe closer back to what
the control was. Now let's say we take that
spiral and we let it grow out and become more linear.
This is translating. It's up to six microns
per second. It's becoming more
stretched out, more linear. Wouldn't you agree?
Yeah. So it looks like
it's getting it's motility back as it's gets more linear.
So the question is, is there some kind of hydrodynamic effect
that we're observing on the flagella.
And that's what we think is happening.
In fact, we think that curvature allows us to control bundling.
That means that if you have something straight,
that's the optimal geometry for the flagella to bundle.
If you start monkeying around the cell and curving it,
the flagella can't find each other as easily.
They don't bundle as well. It's harder for the cell
to translate. So the point here is why
would you want to have lots of flagella
if you can't bundle them. You want to replicate.
You've got to conserve the genotype.
That's what every bacterium is trying to do.
So if you have to put all this energy into flagella and keeping
it moving, that's energy that you cannot put into replicating.
So if you've got lots of flagella, but you're curved,
there's no reason to have those flagella.
They're costing you energy. You're going to lose in the end
because you're going to be out-competed
by another bacterium. That's because flagella
are expensive. You want to get rid of them if
you can't use them effectively. So we ask, have motile cells
evolved shapes that allow them to bundle flagella?
Yes, we think that's the answer I'm trying to get to here.
We think that yes, that is what happens.
And the reason is that we find lots of different genera
of bacteria that have curved shapes, that have only a single
flagellum at the very end. We think that those are examples
of bacteria that may have lost their flagella over time,
as their shape evolved into something that was curved.
So this shows one species, Vibrio cholerae.
But there are 20 different species in this genus
that all have this curved shape with a single flagella.
That is the whole thing there. That is the whole
kit and caboodle. So the conclusion is they're
very sophisticated mechanisms in bacteria.
Bacteria are very sophisticated organisms.
Understanding is just emerging. They have evolved shapes.
Just as we evolve and adapt in response to stress,
bacteria do that. We're trying to understand
why and how they do that. And the take home message
is just keep in mind, I hope that maybe our eyes are opened
a little bit that bacteria are very sophisticated.
Okay, now I'm setting up the next talk.
Part Two. This is cool stuff.
This is swarming. This is kind of the theme here.
What you're looking at, is you're looking at in real time,
bacteria swimming on a surface. Essentially, these bacteria,
these are individual cells, millions and millions
of individual cells that are moving along a surface.
But they're behaving in a coordinated manner.
It's almost like a multi-cellular organism composed
of individual cells of bacteria. There's very rich dynamics.
This is something that we're actively working on.
This is to be continued. But I'll put in just two things
before I end. First, if you're interested
in what life is like at a low Reynolds number,
I brought copies of this paper. This is an awesome paper.
It's a lot of physics, but this is the classic paper
in this area. There are lots of copies,
so feel free to take some and give them to your friends.
And finally, the man who has done a lot of really
pioneering work in this area, his name is Howard Berg.
He's in molecular and cellular biology at Harvard.
He's coming this April to give a talk.
It'll be very hardcore science. It'll be very interesting.
I hope people will feel free to come.
It would be wonderful to see this.
It's in Biochemistry B1118, April 7.
There's the address for the seminar series.
I put that up because the room is probably going to change
because the room that this is in is probably going to be
demolished by April. ( laughter )
Quite possibly. So this is a place you can go
and look at. He's got amazing movies
on bacteria. It's very fun to watch.
Kids think it's pretty cool. At least my kids do.
Maybe they're a little dorky. That's it.
Thanks for coming. It was fun.
( applause ) I'd love to answer questions.
I love questions. Yeah?
( inaudible ) So the way we do that
is using light. Light is, as long as we're above
the defraction limit of light, we're fine.
So using light, or this technique that's called
photolithography, this idea of shining light through a mask,
we can make, practically, structures that are about
one micron. We can actually make structures
that are much smaller than that. But to do that, we have to go
to a smaller wavelength. In fact to do that,
we use an electron beam. We essentially use an electron
beam to write a pattern in the material.
With that, we can get down to around 50 nanometers.
Yes, sir? ( inaudible )
Great question. We're working on that.
Why don't you come to my lab and we'll do it together.
Sorry, the question was, can you make the structure
be more like a three-dimensional object, more like a ball,
rather than just a long, kind of two-dimensional line.
It's not really two-dimensional, because there is some
height there. Yes, there are techniques
that allow you to do that. But they're not really
adaptable to these polymers. So we're trying to do that.
Ultimately what we want to do-- And this is a fantastically
insightful question-- Is not just make bacteria
that have shapes that are long, but make bacteria that have
weird shapes. Like could you take
a rod-shaped bacteria and make it into a cube?
Now you have a cube-shaped bacteria.
There are specific things that we want to study with these.
It's not just the novelty of making a cube-shaped bacteria.
Of course, that would be kind of interesting.
Good question. Yes?
( inaudible ) Immunity?
( inaudible ) With the bacteria
that we're looking at? The question is, can immunity
be seen in the bacteria that we're working with.
Well, these strains were really engineered for laboratory use.
And the strains that we use came out of a human gut
to begin with. The strains that we work with
are not human pathogens. But the mechanisms that we see
in these cells have application to studying pathogenic
organisms. Does that kind of answer
your question. Yes, sir?
What are the flagella made of?
The flagella are made of, most of them, about 99%
of flagella are made of a single protein.
The bacteria, when they make the flagella, it's a very
sophisticated process. They first assemble the motor.
The motor is made out of something like 20 different
proteins that self-assemble into that structure.
Then, the cell actually exports the pieces of the flagella
through the motor. They punch them through
and then they get assembled. So the flagella is actually
a pore, it's like a straw. And it grows by pushing these
proteins through, these things diffuse through the flagellum
and find the place where they get bound
to the next guy in line. Then the next one comes up and
gets bound, and the next one. It essentially forms
kind of a ring like this. But in each flagellum,
there are roughly 10,000 copies of this protein.
It's all protein. Yes, sir?
( inaudible ) Okay, so the question is,
why would you have curved bacteria at all, because
maybe evolutionary pressures are selecting toward rods.
It's a great question. In fact, I'll be honest
with you. I don't even think we know what
bacterium necessarily look like when they're in the wild.
Many of them probably have the same shape that they do
in the lab, but we don't know how to mimic the conditions
for growing bacteria in the wild.
That's why something like 99.99% of all microbial strains
can't be grown in the lab. We don't even know what they
are, because we don't know how to grow them.
And I don't think we know what they look like.
So I don't know the answer to your question.
The question is, are the shapes that we're seeing in the lab
really representative of what bacteria really
look like in the wild. That's "A".
"B" is, what kinds of pressures have driven them
to their shapes. And I think, I don't know.
Here's an example of where, you know, this is a shape that
you might not have expected. But you can take a swimming cell
and not just have a rod, but it turns out a swimming cell
can have any shape. In fact, there are swimming
cells that are perfectly flat, square plates.
And they swim very well in liquids.
So it's really a wonderful question.
I'll tell you next time. Yes, sir?
I imagine there's an enzyme that breaks it down.
That's right. >> So that you have like a--
( inaudible ) But when does that occur?
Good question. So it must be, because the cell,
the growth of the cell has to be somehow coordinated with the
replication of the chromosome. Because you don't want
to grow faster than you can make a copy of your DNA.
Because if you do, then boom, you just close off that cell,
and the daughter cell is floating away
and it doesn't have any DNA. It's dead.
So the growth, the decomposition of the wall must be
somehow timed to replication. And actually, the decomposition
of a wall must somehow be coordinated with insertion
of the wall, too. It's very complicated.
There's actually a whole class. There's something like
12 different enzymes that people know are involved
that are called the Penicillin-binding proteins.
Those are the targets for all the known
beta lactam antibiotics. There's finally a crystal
structure of one of those proteins, just this last year.
It was a huge deal. But that's the first one of 12.
Maybe there are even more of them out there.
So we have really no idea. Yes, sir?
Do all bacteria have flagella?
No, not all bacteria. Some bacteria just rely
on free diffusion. Some bacteria have little
structures that allow them to stick on the surface, so that
nutrients can flow past them. There are all kinds
of different mechanisms. Some actually flap
back and forth. There's a whole variety.
And there are cases where there are not any.
Yes, sir? >> Do you consider things
like the surface area of the bacteria--
( inaudible ) And another issue is, I think
there is evidence that a lot of bacteria populations have
been somewhat controlled by the neighboring bacteria.
That could be another factor in shape.
So the first question was, does surface area matter.
And it surely does, because you've got to import stuff
across your surface area. But there's a balance
between size and surface area. I mean, you know, you get big,
you have a lot of surface area. But if you get big,
it costs you more energy. You have to spend energy.
Maybe shapes have been selected to maximize surface area.
That's very possible. In certain conditions.
I would be amazed if that's not the case.
The second question is, maybe the variety of shapes
has to do with communication between bacteria.
And it's clear that bacteria are able to communicate
with small molecules that act as signals to turn on and off
transcription of genes. And that maybe this kind of
cross talk between cells plays a role in cell shape.
It's very possible. Yes, sir?
You said that the flagella are on at all times.
Yeah. >> But in the lab, you
put them in various solutions, how do they get nutrients?
That's a really good question.
So there are conditions that if you grow cells--
There are conditions that you can grow cell under in which
they will not express flagella. That's usually--
Julius, is that in very, very rich conditions?
In that case, they won't actually express flagella.
But you know, even with media that has less nutrients,
in which we'd still think there was an abundance,
where you really wouldn't have to move, because diffusion
is passing things over you very quickly, those cells
still express flagella. It's a good question.
( inaudible ) So maybe flagella
have another function. ( inaudible )
That's a great example. We know that when these cells
that I showed you-- Well we don't know this,
but we're working on it. But in this case, the flagella,
we think, are actually allowing the cells to coordinate
their movement. So flagella also allow cells
to stick on surfaces. They're probably
other unknown uses. It turns out that there's
a short organelle and bacteria called pillae.
Pillae are used for essentially, sex, allowing
bacteria to exchange DNA. These pillae, oddly enough,
in the last couple of years, people have found that they
are very electrically conductive.
It's unclear. That's not in every species.
But in some, they're very conductive.
So the question is why would you need to have
conductive structures. Maybe it wasn't programmed.
Maybe it just happens to be the way that it is.
Maybe it does have a function. Yes, sir?
Do these motors all rotate in one direction?
Can you tell if they rotate all counterclockwise?
So the question is, do the motors all rotate the same way.
And the answer is yes, when the cell is running,
if you look from the back of the cell, all the motors are
turning the flagella this way. That causes the cell
to actually rotate this way. The way you can do these
measurements is you can take a cell
and engineer the flagella. And this is Howard Berg's work,
beautiful stuff. Where you stick a cell down by
it's flagella and then you just watch the thing rotate.
And it rotates very fast, so you have to have a very fast camera.
You can actually measure the switching of the motor.
And you can measure. Imagine if you take
a big bacteria, it's going to have more drag.
So you can actually calculate, you know, the mechanical
properties of the motor under different loads
and efficiencies. He's done all this work.
It's very beautiful. >> Like a motorboat propeller,
can you put it in reverse and go backwards?
No, the bacterium never does that.
There is a bacterium though, it's very interesting.
As it moves, the flagella are bundled.
And the cell swims down and hits a surface,
then the flagella all go to this side
and the cell moves this way. So it's capable of flipflopping.
But there are no bacteria I know that do that, in reverse.
A paramecium does that, but you know, a paramecium
is not a bacteria. ( inaudible )
The body is rotating, yes. You know, you've got
to balance the forces. There's a balance of forces
in everything. If the flagella
are rotating this way, it creates a torque on the cell,
so the cell body has got to rotate this way.
The cell is essentially doing this.
( inaudible ) I don't know off the top of
my head, but there's a paper that has all those details
of the motor. It's very efficient.
So what's a combustion engine efficiency?
It's about 30? I was going to say like 20s.
I think E. coli is like double that or even better.
So it's very efficient. ( inaudible )
Probably some. I mean, the question is,
how can you move an ion through something
and get it to rotate. That's huge.
When someone figures that out, that'll be a phenomenal story.
And how do you get a motor to switch
and go back the other way? Yes, sir?
( inaudible ) Interesting.
So, no, but there are organisms that are on the same size scale
as a bacterium that do that. And one of those--
Oh, yes, sorry. The question was, are there
bacteria that don't rely on the rotation of the flagella,
but instead use a mechanism more like a squid,
or use multiple flagella in a different fashion to move.
And there are bacteria that kind of whip, are capable
of moving by whipping. There's a bacterium that,
they're either really rotating or whipping, but there is one
that fits your criteria. It's not a bacterium though.
It's an algae. That organism
is very interesting. It takes two flagella,
it essentially has arms, and it does a breast stroke.
It literally does a breast stroke.
It's not quite like a squid, but it's getting kind of
towards what you were asking. And it's fantastic, dad.
You can get this stuff, you can order it for like a buck.
And you can grow it under light. It doesn't need anything.
It's wonderful to watch. Yes, sir?
( inaudible ) That's a fantastic question.
The life span of bacteria. What do you think
a life span is? Wouldn't the cell
just replicate forever? If you just kept
feeding it nutrients forever, couldn't it replicate forever?
Well, it turns out that it looks like bacteria do age.
And the way you can tell is that you can distinguish the
mother from all the daughters. And the way you do that is the
mother cell, every time a cell replicates, it's got
to make all these proteins, all these genes and chromosomes
are being turned on, transcribed,
translated into proteins. The proteins do whatever
their role is in the cell. Sometimes the proteins misfold,
they misbehave. And so when those misbehave,
the cell needs to get those out of the system,
because they could be toxic. So the cell puts them into
what's called an inclusion body. And the inclusion body
gets tacked to the end of the cell, the pole.
Over time, that inclusion body accumulates more and more
protein. So what you can do,
is you can put protein that misfolds frequently
and on the gene, you can put another gene that expresses
a fluorescent protein, that's from a jelly fish.
So every time that protein is produced, it's got a
fluorescent molecule attached to it.
You can measure how the fluorescence accumulates
over time. So the mother is going to have
much more fluorescence, because it has more
misfolded protein than cell number 1,236,512.
So that's kind of what we think about in terms of aging.
There may be other molecular mechanisms that are effective,
besides just protein. But it looks like cells,
bacteria do have something that we could say resembles aging.
Yes, sir? >> What's an E. coli chromosome?
Is it pure DNA? >> Yes, so the chromosome,
it's a piece of DNA. It's circular.
There's something like 4,500 genes in E. coli.
So you assume there's 1,000 bases per gene,
so there's something like five million bases.
Five mega bases, something like that.
So it's a big, circular piece of DNA.
It's not just a free-floating circle.
That would be enormous. It's got to be packed up
really tightly. And so, it really occupies a lot
of the evolvement of the cell. It's just kind of
squished in there. ( inaudible )
So there's this place called origin, which is where
the replication starts. And the DNA has to essentially
get folded back a little bit. This mystery is, how does
the chromosome get away from the new chromosome?
Because now you have one massively complicated pile
of string, and then you have another massively complicated
pile of string. And you kind of, you know,
do this with two pieces of circular string, and then
try to pull them apart. It's going to take you forever.
How does the cell get that other piece of DNA to the other side?
Now we know in eukaryotes, there's this mitotic machinery.
The chromosomes line up, and there are microtubules
that grab on, essentially, and pull them to the poles
and the cell divides. That doesn't happen in bacteria.
So how does that happen? I don't know.
Maybe someone here will figure it out.
Yes, ma'am? ( inaudible )
Oh, yeah-- The question is,
so sorry about that. I'm terrible about that.
Have there been any bacteria that have become extinct?
Quite certainly. As bacteria evolved,
there came a point in time when bacteria "X", you know,
it just ended up having this really strange,
very energy-consuming structure that just could not
deal with some stress. And it got out-competed by
all the other bacteria that were looking for the same resources,
and it went extinct. You can trace through lineages.
Undoubtedly, there are billions of strains of bacteria that
have probably gone long ago. Yes, sir?
When did they first discover this E. coli, that everybody
gets all worried about? >> When did they discover it?
I think it was in Japan, ten years ago.
( inaudible ) I mean, it was probably around.
It was probably around, and it was just under those
certain circumstances that it proliferated
and caused a problem. I don't know.
That's not my area of expertise. But it's possible.
( inaudible ) Oh, really?
( inaudible ) So if you do like sequencing,
it turns out that it's not related to E. coli?
( inaudible ) Oh, I see, okay.
Any more questions? ( inaudible )
( laughter ) Right, they're very warm
and friendly. Never cause any problems.
Doing you a lot of good. As you know, after you take
antibiotics, it's miserable for a couple of weeks until you
get your system back in swing. ( inaudible )
Thanks. ( applause )