117 Doug Weibel

(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)