9UNP0173SD_Katrina_Forest_WordPress

- Welcome, everybody, to Wednesday Nite at the Lab. My name is Tom Zinnen. I work at the UW-Madison Biology Center. I also work for UW-Extension Cooperative Extension. And on behalf of the Wisconsin Alumni Association, I would like to welcome you to Wednesday Nite at the Lab. We do this every Wednesday night, 50 times a year. And we also appreciate Wisconsin Public Television taping these and broadcasting them later. Tonight, we have professor Katrina Forest, who is an associate professor in the Bacteriology Department at the University of Wisconsin-Madison. She's talking about "Bacterial Infections in Three Dimensions." 

I have to pause here for a personal announcement. My dad had a 12-centimeter infection right here in his right biceps. The abscess was the size of a golf ball. He lives in Illinois. I think the Department of Conservation in the state of Illinois is considering putting a marina in there. It is the best example I've ever seen of what bacteria can do to you. And I'm looking forward to seeing if you have any pictures to match the one I took. She received her BS degree in Applied Biology from MIT, and PhD in Molecular Biology from Princeton University. And in addition to her research program on the Structural Biology of Pathogenesis, Dr. Forest teaches a class entitled Microbiology and Atomic Resolution. 

The goal of this is to help microbiology students become confident in assessing structural biology literature. She is the director of the UW-Madison Women in Science and Engineering Residential College. She serves on the steering committees of the Biophysics and Biotechnology training programs. Please join me in welcoming Professor Forest to Wednesday Nite at the Lab. 
(applause)
 
- Thank you very much. So I've been following, for a long time, the syllabus of Wednesday Nite at the Lab. I think it's fantastic. And I was really interested to get your mail with the instructions from the regular visitors about how should comport myself tonight. 

And I understand that there are going to be lots of interruptions, which I welcome, so that's fine. I'm going to give you a whirlwind tour of some of the things about microbiology which I find the most fascinating. And the title is kind of a conglomerate of the things I want to talk about. First I'll focus on this part about infection. And I'm going to try to convince you that although I understand that infections can be bad, many of the times your interactions with microbes are actually extremely beneficial. Then I'll talk about a few examples of microbial structures that we need for these interactions, or they need for these interactions. I'll tell you a little bit about the technology of protein crystallography that we use to study the structures. And then at the end, I'll tell you a little bit about the specifics of the results from our lab. So these are two of my favorite humans, my husband and my daughter at the Birkebeiner. And the scale here is about a meter. 

And on the right-hand side here is the favorite microbe of many microbiologists. This is a single E. coli cell. And very much, molecular biology and really important research on the central dogma of biology has been done by studying this micro-organism. This is about one micron. So the difference in scale between a meter and a micron, we have a meter and one tenth to the minus six meters. So we can get the difference in scale here. Now, I know that you already know that there are a large number of microbes associated with you. So that shouldn't be a surprise. But I wonder if you have some idea of the absolute magnitude of cells that you have in your body, versus how many microbes you have in your body. 

So how many cells, human cells, do you think there are in your body? Raise your hand if you think there are more than ten. 
(laughter)
 Okay, keep your hand up if you think there are more than 100. Okay, how about more than 100,000? How about more than 100 million? Any ideas? Okay. So actually it's way more than that. There are about ten to the 13th, or ten to the 14th human cells in your body. 

So that's, I mean, I know you all know this. That's one with 13 zeros. Well, okay, maybe one more. 
(laughter)
 Right? Roughly speaking. Okay. Now how many bacterial cells do you think there are in your body? This is the number of human cells you have. Raise your hand if you think it's more than ten. 

More than 100. More than 1,000. More than a million. More than a billion. More than a trillion. You guys are smart. It's this number plus a zero. So there are ten times more bacterial cells in your body right now, than human cells in your body right now. So when we think about the human genome, imagine, you know, actually a lot of that DNA is not human in origin. It's microbial in origin. 

And this is a forefront, I think, of microbiology and actually, largely, of human medicine these days, to understand how your health and development is affected by those bacteria that live with you all the time. So the human body is a home to something like 10 to the 13th, or 10 to the 14th microbes. And if you come away with one take-home message tonight, it's that there are about ten times as many microbial cells in a healthy human body than human cells. Humans are born sterile, but instantly begin their lifelong association with microbes. And actually, the proper development of the human, including in particular the immune system, the gut immune system, requires those microbes. So there are, I just told you, 10 to the approximately 14th, or 15th microbial cells in you. And these comprise something like 2,000 to 3,000 different species of bacterial cells. Most of these are in the gut. So a lot of what we understand about microbiology in humans has to do with gut microbes. But there are also microbes that protect your skin from infection by pathogenic microbes. 

Your mouth is absolutely full of very interesting complex microbial consortia. And basically, most of your mucous surfaces are also home to a very complicated microbial community. Of those 2,000 to 3,000 microbes, only 80 have ever been shown to be human pathogens. So I absolutely don't want to, you know, under-emphasize the critical nature of pathogenic infections with humans. Obviously, that's why that's where we are and how we learn most of what we know about microbes so far, by studying diseases and trying to control them. But just on the absolute scale of, you know, who is bad and who is good, I think it's important to understand these numbers. Most microbes are not dangerous. Most of them are good for you. Of course, some are bad for you. And this is a list of some of the ones that we know that cause disease in humans. 

So I've kind of said some of this already, but your normal flora protect you against infection by microbes, which might otherwise be dangerous. So this is true of a lot of skin diseases, for example. They stimulate the development of the immune system and the continued health of the immune system. In particular, this lymphoid tissue part of the immune system. The microbes in your gut make many vitamins for you. In particular, Vitamin K, the only source you have of Vitamin K is the gut microbes who make it for you. As well as some B vitamins. And I put this up here as sort of a nod towards the idea of probiotics, which for a long time, I think there was general understanding that probiotics had some sense of real science behind it. But actually, there's a lot of really interesting, bona fide science now trying to study the molecular mechanisms of how probiotic bacteria protect you against infectious bacteria, which is kind of interesting. So most microbes, most bacteria, and I'm talking about bacteria that, for those of you who are specialists, when I say micro, that means bacteria and -- live not in isolation, although you see a lot of pictures and you learn a lot in science labs about individual bacteria swimming around in solution. 

In fact, most of them live in complex communities with one another, both in the environment, this is just a photograph of the top of sort of pond scum. These are microbes living in communities with each other. And this one is a little more scary. This is a catheter in a hospital. And they are also coated very often with biofilms. That's this name we ascribe to microbes that live in communities together. And they are actually sometimes required shared nutrients among the microbes in a community. Sometimes shared chemical reactions, where one chemical reaction pathway over all is energetically favorable, but no one microbe can carry out all the steps in the pathway, so they share reactions and products, and derive chemistry in the environment. And the structures also are often very complex and tightly interacting microbes sharing soluble nutrients with each other and soluble signaling molecules to initiate transcription pathways, and gene expression pathways, and developmental pathways. In your body, where do you think there might be a biofilm? 

Right, absolutely. So, teeth is another really complex biofilm. This is a very good example of a case where, if you kill all the microbes in your mouth and the wrong ones colonize first, that can lead to tooth decay. So having the right microbes in the right order in the mouth is actually a very important part of good oral health. That's an important one. So there are now genome projects looking not at the genomes of individual organisms, but for example at the genomic content of all the organisms in the mouth, to understand who, in the anthropomorphic sense, is in there, even if we can't grow these individual organisms in the lab. So I'm going to dive in a little further to a somewhat higher resolution conversation and tell you that biofilm formation requires several very specific appendages on the outsides of bacteria that they use to attach to solid surfaces, and to interact with each other, and also for motility. Let me just give you two examples. One is flagella. So this is three bacteria with these long hairs sticking out from them. 

Flagella are very famous, because they propel motion in many swimming bacteria. And they do this by spinning in one direction, they drive bacteria forward. And that moves them usually up a chemical gradient toward an attractant, or down a chemical gradient away from a repellent. The way they switch directions is not very well planned on their part. They actually tumble, switch directions randomly by reorienting the direction of rotation of the flagella. So flagella spinning one way drive the bacteria forward. Flagella spinning the other way cause them to tumble. And what they do is decide if they can sense a gradient, if they're going in a favorable direction, they don't tumble. If they're not sensing that they're going in a favored direction, they tumble and reorient. So this is a very random motion, except that it's biased by staying in the direction that's favorable if they can sense that gradient. 

So these structures have been studied in incredible detail by a lot of high resolution technologies over the last 50 years. And I want to show you an amazing movie. This is from the lab where they've done a lot of high resolution structural work on the flagella. This shows the assembly of the flagellar base inside the membrane of a single bacterium. It assembles with dozens of individual protein sub-units that make a rotating motor, with a bushing and motor that's driven by a proton gradient across the cell membrane. There's an extremely well-ordered linear progression of the assembly with various adapter proteins after a certain number of hook subunits get added. And then finally, the major subunit is actually secreted out the flagella and added on at the tips. So, thousands of copies of the individual flagella proteins are added on, one at a time. And what I find amazing is that they're actually secreted up through the hollow filament. So after thousands of those have added on, you get these flagella which are many times longer than the individual bacterial cells. 

And they rotate. And that's what drives that. That's the base of the flagella motor. Those are fluorescent images of real bacteria with their flagella behind them, rotating and propelling them through liquid. That rotation and that swimming, and also just the attachment that the flagella allow is important for biofilm formation in many cases. So another filamental structure on the surface of bacteria that they use for motion, but on solid surfaces, are these. These are called pili. At this resolution, they look a lot like flagella except they're straight. And instead of twirling, they come in and out of the cell. This is a fluorescent image of a single bacterial cell with the filamentous pili, which just means hair in Latin, that are being assembled and then retracted back into the cell. 

In this particular image, the cell is stuck to a glass cover slip. So the cell isn't going anywhere. It's throwing out pili and reeling them back in again. In the case where the cell is not stuck. Yeah? 
(inaudible)
 It's about ten-fold faster than real time. So the retraction rate is about a micron per second. And those cells are about one micron long. But if the cell is free and the pilis is what is stuck to a solid surface, then what happens is that the pilis sticks. 

So now it's going very fast and looping over and over again. You can see this one cell and the tip of the pili stick. And then the cell gets pulled across the solid surface instead of the filament being pulled back into the cell. And that's what allows these cells to crawl over these solid surfaces, and to grab onto each other and pull themselves into a more tightly knit community. So I think of this, these are the structures in purified, sheared off of bacterial cells. These are just electron micrographs with the base. Whoops, here we go. This is the base of the pili after they've been ripped out of the membrane. So, we would like to understand how these kinds of structures work at a molecular level. And we think that in order to understand how they work, we should really understand their structures. 

The idea being that the structures probably have something very closely related to do with the function. This is the idea that form follows function. The Sydney Opera House, and everything inside it, works as well as it does because it was designed properly. And its structure is exactly what you need to hear beautiful opera. Our goal is to understand the structures of some of these bacterial appendages, in order to ultimately understand how they work, and how they mediate both beneficial interactions as well as pathogenic interactions of bacteria with humans. So this is a little bit of a review for those of you who are already biochemists, but maybe something new for those of you who haven't had biochemistry yet. Those structures that I showed you, the flagella proteins and the pilis proteins, are all made up of the same polymers of 20 different amino acids. The proteins that you eat in your diet and the proteins that carry out almost all the enzymatic reactions in your body are all made up of linear chains of 20 different amino acids. And each protein is sort of on the order of a few hundred amino acids long, a hundred to a thousand, roughly, is the length of most of the proteins in your body or protein in a bacterium. We all share the same code of 20 amino acids. 

Each amino acid has the same chemical structure mostly. The black atoms are carbons, oxygens are red, nitrogens are blue and hydrogens are white. And what changes among the 20 amino acids are the groups that are stuck on in these red R's. So there are 20 different variations of R, but the general chemical structure of the backbone is the same for all of them. And one thing that's very important that gives proteins some of their characteristic folded structures is the fact that this bond is planar. So in every one of these peptides, there is one bond around which rotation is very difficult to accomplish. And that limits the confirmations that polypeptide chains can adopt. So because of this set of shared atoms all the way along the peptide backbone, there are some predictable interactions that happen regularly in many different proteins with structures we know. Those include the alpha helix, which was predicted long before its structure was solved. And in this situation, the amino acids adopt the helical array where the nitrogens and the oxygens from amino acids up the helix interact with each other by sharing a hydrogen. 

This is called a hydrogen bond. So, oxygens that are attached down here share a hydrogen up the helix with this nitrogen. That really stabilizes this helix. And we see this structure over and over again in protein folds. Another common secondary structure that is dictated by this set of atoms, which is the same in every amino acid, is again, a sharing of a hydrogen between an oxygen that's attached to a carbon and this nitrogen. And these give us, we call them pleated beta sheets. So this cartoon sort of illustrates the trace of the peptide backbone. And here, one backbone comes here and comes around and comes back down. And they share this hydrogen across the strands to stabilize the structure. So alpha helices and beta sheets are very regular structures inside proteins. 

And we would like to know for any one protein what its fold looks like. Because in many cases it's really helped us to understand the biology of how a protein works. The trouble is that these bonds are on the order of ten to the minus tenth meters. So instead of having ten zeros after this for a long number, we have ten before it and a decimal point, so they're very, very short length protein bonds. And we can't study those using light microscopy, because the length of light is too long to give us information about the spacings between these atoms. We need a different kind of radiation that has a shorter wavelength. So we use x-rays, which have wavelengths that are on the same order as the spacing of those atoms. In order to use x-rays and get enough signal to learn something about the proteins, we need thousands of copies of the protein all in the same orientation. And so in order to do that, we've crystallized proteins. So in the same way you can leave a solution of salt or sugar out on your kitchen table and evaporate the water and end up with crystals, we can do a similar thing with protein molecules. 

We get beautiful crystals in the lab. Some of these are really good for collecting data on, the large single ones. And some of them are not, but they are still pretty. These are snowflake crystals that are useless for collecting data in the lab. All of these are crystals that we grew in my lab over the last few years. And nice crystals, we put in an x-ray beam. And x-rays are impinged on the crystal and we get diffraction like this. Each of these spots is from the coherent interaction of scattered x-rays. I don't want to spend too much time on how we grow the crystals. But it's just chemistry. 

These are phase diagrams like any other chemical process. We have a protein in solution. And we add to it some kind of precipitating agent that increases the chemical potential and drives this protein out of solution. The trick is to take this protein out of solution as a stable crystal, rather than as a precipitant. So, if you fry and egg in the morning, the protein that comes out of solution in your fried egg is no longer folded the way it would have been in a cell. And it is no longer functional. And it won't do us any good, really, to solve the structure of that kind of protein. So we want to coax these protein molecules to come out of solution in a way where they're still functional, but extremely concentrated and crystalline. We play around with the conditions and solution until that happens. And at a practical level, we do this by taking these plastic trays, 24-well trays. 

And in the well, we put a solution of salt or organic solvent like this one, this reservoir. And we actually hang little tiny, four microliter usually, drops of protein solution in vapor equilibrium with this mother liquor, and seal it up here at the top with grease. So this is a closed system. And what happens is the concentration down here in this reservoir of salt is higher than up here in the drop. And that's not a stable situation. And so in order to equilibrate this experiment, since the protein is not volatile and it can't move across the vapor phase, water leaves the protein drop and moves across the vapor phase into the reservoir until these are equilibrated. And what happens, is this concentrates the protein in the drop. If it works, then the protein comes out of the solution as a crystal. Then we put the crystal in an x-ray beam. This is just a picture. 

There is a little crystal. I should have brought one of these, but I didn't. These are little, tiny essentially like soap bubble loops. If you dip a soap bubble loop in film and blow it, you know, you get surface tension holds the film in there. We hold our crystals in little, tiny soap bubble film loops, and shoot x-rays from one direction. Then from an orthogonal direction, we blow very cold nitrogen gas at the crystal and keep it at 100 degrees Kelvin in order to prevent radiation damage while we're doing the experiment. So here is a real-life example of a crystal from our lab. This is really a green protein. It's a light receptor from bacteria. And this is just a UV visible spectrum of the crystal, which is simply to show that crystalline proteins have the same properties as proteins in solution. 

So even in a crystal, there is still about 50% water. Most of the time, chemical reactions happen in crystals. And this is used as evidence that the structures we saw from crystalline proteins are relevant to what happens in cells. Then this is the kind of information we get after doing this kind of diffraction experiment. We get blobs of electron density. They're almost meaningless unless you really kind of know enough about protein structure to trace into these blobs, some atoms. This is the structure of that photo receptor protein. Every one of these yellow corners is a carbon. The red ones are oxygen. The blue ones are nitrogen. 

This shows an example of one of those beta pleated sheets that I showed you in the cartoon. They're fit into the electron density from a crystal of a protein that we purified in the lab. This is still pretty blobby. I don't really think you can see exactly where each atom is. The only way we can place them in there is because we know something about protein structures to start with. We sort of know what the choices might be. But there are some proteins, this is one that binds to sugars. It is also from a bacterium. They crystallize extremely well in beautifully ordered arrays, and we get much stronger diffraction data. This, again, is a diffraction experiment. 

But the information is much denser in this kind of picture. We have many more of these diffraction spots. And we can get a much more well-defined structure. This is the same idea. This is a different protein. The same kind of experimental electron density map, but it contains much more information because the crystals are much more well-ordered. And now, you really can say there is an atom there, an atom there, an atom there. And you can really trace through the structure. In fact, you can even see the difference between, for example, this is a carbon and this is an oxygen. You can tell that oxygen has a higher molecular mass than carbon, because even in this electron density map, it's larger. 

This is beautiful, high-resolution electron density. From this kind of data and refinements, we get structures of proteins. So this is a protein from a bacterium. It's actually from E. coli, that one I showed you at the beginning. And it binds glucose right here in this cleft. And without glucose, it's an open structure. And with glucose, it's closed. And the difference between those two confirmations allows the bacteria to tell that it's headed in the right direction toward glucose. This molecule is actually important for that flagellar motion that I talked about at the beginning. 

In this sort of high resolution structure, we can really determine with extremely good accuracy, exactly where each atom is and what the distance is in units of 10 to the minus 10th meters R, between atoms. And this is important for things like drug design, where these kinds of distances really do have an impact on binding affinities. Okay, so coming back at the end to pili. This is a picture of a single cell with the type four pili hanging off the end and a single flagellum. So this is an organism that has both pili and flagella. And my lab is particularly interested. Our real focus is on the assembly and retraction of these filaments. We have solved, over the years, the protein crystal structure of quite a few of the subunits from different species that make up these pilis filaments. And these are just some examples. So the organism that causes Gonorrhea. 

And the organism that causes a lot of infections in immuno compromised people in burn victims, in Cystic Fibrosis patients. This is an organism that's ubiquitous and doesn't usually bother us unless we're compromised somehow. So the point here is that each one of these species, although they have slight variations in the structure which lead to differences in binding specificities, for example, they all have the same underlying architecture, this long alpha helix and this beta sheet at the end. And this architecture allows these proteins to associate with each other. So these long alpha helices pack in the interior of that long filament, and we get these beautiful filaments that we now have quite high resolution information, largely from the lab of Lisa Craig, that shows how the whole filament assembles together. And we understand a lot about what's exposed on the outside of the filaments, and what it might take to interrupt the assembly of these filaments, for example. A lot of work has gone into the idea that the proteins from these filaments might make useful vaccine candidates. Because if your body has antibodies against the filaments they won't be useful to the organism for attaching to surfaces in your body. The other thing we've been studying lately, and actually Ken is here, who's done a lot of work that leads up to what I'm about to tell you, is to study the mechanism of the motor retraction. I showed you the movies of how pili retracted into cells and mediate motion. 

And I really think that this is the Ernest Hemingway vision of fishing, where you know, you throw out a hook and you catch a fish which is too big to handle. And you reel, and reel, and reel, but what happens is you go into the ocean instead of pulling the fish into your boat. So we're studying the motor mechanism of this retraction inside the cell. So that motor is called PilT. When we purify it from gonorrhea, we see these donuts. This is freeze etch electron microscopy. And we also know from other data in the lab using purified proteins, that these donuts are made up of six proteins, six identical PilT proteins. This is the motor that does that retraction. So Anna, in our lab, crystallized this motor protein. And we solved the structure and found a protein which has all the hallmarks of a classic protein fold. 

It has beta strands, alpha helices. And it's got ATP, which is the source of energy for the motor motion, found in this cleft between the two halves of the protein. This is how the six protein subunits align with each other in this donut shape. And within each donut, what we find is that the orientation of the top half of the protein with respect to the bottom half of the protein changes. So we have a sort of a Pac-Man motion going on in this PilT retraction motor. Anna has made a movie of how she thinks this may work in a cell. The idea is that each one of these colors is a different individual protein subunit. When our small molecule binds in the cleft between the two halves of the protein, and causes it to clamp together. The protein on the right-hand side pushes on the protein on the left-hand side. And this generates force around the motor. 

So we actually have a long, long, long way to go, but two possibilities for how this force generation might work within the cell. This is a very schematic depiction of the inner membrane, actually, of a bacterium with the pilis assembling out into the solution and the motor on the inside. So one possibility is that these PacMan motions of each PilT subunit are pulling directly on the pili and retracting them back into the cell, in a simple hand-over-hand motion. Another possibility, which we're also investigating is that the motor PilT doesn't actually touch the filament directly. Perhaps what the motor does is open up a channel in the membrane by pulling on other proteins. And this allows the filament to retract somewhat more spontaneously back into the cell. So these are the sorts of mechanisms that we're trying to distinguish between. This is just to remind you how cool this is. I like to just watch this movie, because that's really what we're trying to understand, how that retraction happens at a molecular level. Blocking that retraction, even if you don't kill the cell, prevents biofilm formation, and might actually inhibit the interactions of humans and these microbes. 

So I would like to just acknowledge the people in my lab who have done all the work I talked about at the end here. Especially Ken who is in the audience, who solved the first PilT structure. And Anna, who solved the more recent one that I showed you. And I would also like to acknowledge funding from the NIH and the Keck Foundation. And I would like to thank all of you for your attention. I'd be happy to take questions. 
(applause)
 Yeah? 
- When the pili is retracted, is it disassembled? 
- Yes, we believe it's disassembled. 

In fact, I meant to pass these around. I'll interrupt myself for a second to pass these around. This is the flagellar motor base. I will pass this around for anybody who would like to look at it. And then, yes. So this is a pilin subunit. This part of that alpha helix is very greasy. So what we think happens is, as the pili are retracted into the membrane, which is also very greasy, we think that a very stable sort of holding pattern is for the individual pilin subunits to be disassemble and just be stored in the membrane. Yes, exactly. This is a model of five pilins sort of assembled with each other, with their long helices sticking into the membrane, which I can also pass around. 

And this one is that motor protein. You can see the six subunits. Each one is a different color. And one of them is shown as this sort of trace of helices. And the others are shown as the volume that each atom would actually take up. I'll start this one here. This one and the flagella body were printed here in this building on a three-dimensional printer that we have, which is a lot of fun for studying three dimensional protein structures. Other questions? Yeah? 
- The two models that you showed, the one where hand-over-hand, and second one, why does the pili not just leave instead of being absorbed? 

- Oh, well, that's a good question, so you could imagine a lot of possibilities. One is because of this idea of the aqueous versus lipid phase. So if they're only stable in some kind of lipid phase, that would drive them back into the membrane rather than being released. The other possibility could be that there is a protein inside the cell with some affinity for the pilin subunits. And that it is essentially, it may not be active. It may just be that it binds them. So in a sort of a hit or miss action, if it gets pulled in, they're more likely to be held there, because of some interaction with the cellular protein on the inside. 
- Back in the day, I thought the pili was for natural exchangers and genetic. 
- That's a great question. Pili is a very general term. 

It actually just means hair. And there are lots of different kinds of pili. So there are conjugation pili. And you're absolutely right. Their role seems to be to help bacteria exchange DNA with one another. And there are pili whose sole role is for attachment, which don't do this kind of retraction. And then there are these pili which really sort of make their living by doing this retraction. Yeah, you're right. 
- You showed us the retraction of the pili. What about the extension? 

Are you studying that part of it? 
- Well, you can't study one without the other in a sense. So actually, yes, we are trying to crystallize the motor that does the extension. It's very similar in sequence and in structure to the motor that does the retraction. So actually I think, you know, there may be some sense in which the motor interacts with the same base proteins. And depending on which motor is there or which switch gets flipped, you know, you may either extend or retract the same subunits. 
- What kind of time frame for extension versus retraction? Retraction is very quick. 
- It's about the same. It's very similar. 

One thing that's fascinating, I think, is that the retraction is very processive, which means once it starts, you don't tend to see jerks. You don't see a lot of starting, stopping, going out or coming in. It tends to be, once a pilis retraction begins, it goes all the way to completion, which I think probably tells us something about the mechanism. Yeah? 
- So presumably, this is not driven by new protein synthesis. 
- That's right. That's a good question. The question is, could this be driven by new protein synthesis. If you do a back of the envelope calculation, and think about all the protein synthesis that's happening in a cell at a given time, you could just about make it work, if all of your protein synthesis was going to making new subunits. So it's not off by orders of magnitude. 

But it wouldn't make very much sense. The real evidence that subunits are recycled is kind of scant. But the evidence that there is, is that fluorescently labeled pili can be retracted, and then when new filaments are made, even though the fluorescent dye is gone, they're already fluorescent. So the thought then is that the subunits were already labeled before they were retracted and then being reassembled. 
- A pH gradient is part of the motivating force for this? 
- Yes, so there isn't much. Well, these bacteria, most of them, at least, okay. So, it's a good question. The question is, could it be a pH gradient which drives the disassembly and assembly. First of all, proton motive force, which isn't the same, but isn't required. 

So, you can block a proton motive force and still get retraction. There are enough bacteria which live at neutral pH and have the same pH, both inside and outside the cell, that are able to do retraction, that that seems unlikely. There are also bacteria that live in extreme pHs and have different internal and external pHs which do this. But since there are some that aren't like that, I don't think that's the answer. Tom? 
- A couple of questions. How much amount of that energy? How many calories do you burn up an hour doing this? If they're starving, do they do this? And the other is, on a new analogy, if you have a rope you can throw it out. 

If you've got an anchor, you can pull yourself forward. 
- Right. 
- If you've got a big, long pole, you push yourself. Are there examples of both those things that are flexible? 
- Sure, sure, sure. So, to answer the first question, which I already forgot. It was, how much ATP does it take to do this, how many calories do you burn. Believe it or not, we don't know the answer. The reason we don't know the answer, which seems silly, is that because this is a pretty complex process which goes through two membranes and involves a couple dozen proteins, we can't do this in vitro yet. We can only do it in whole cells. 

We don't have a way to measure how many ATPs are turned over per subunit retraction in isolation. 
(inaudible)
 
(laughter)
 So that is actually a project. Lorraine Meyer in my lab is trying to create a sort of an artificial system where we express the minimal number of proteins that we absolutely need for the assembly and disassembly. Get rid of all the regulators and all the other bells and whistles. And then try to put the system into vesicles that are isolated from live cells, so that we can start to ask those very specific sort of biophysical questions. I'm afraid I can't answer that. And your second question about pushing versus pulling, assembly of these pili does not push cells. 

There is not enough tension, because they're too flexible. There are motility systems in bacteria. And this is kind of hotly debated whether this is really what happens. But there is this idea that there are nozzles on the backs of some bacteria, that slime is just squirted out the back of, that propels them forward across the solid surface. It's the fire hose hypothesis, basically. Questions? Yeah? 
- The general purpose of microbiology, in the early part you talked about the ten to 100 times more microbial cells than human cells. If you take a broad spectrum antibiotic for an illness, what kind of setback does it give? 
- That's a great question. 

And I don't. It's also a caution, right? Just killing all your microbes is not necessarily something you want to do regularly. And I have no idea what the answer to your question is. 
- There are some good examples of it. One of them is if you have a patient who has been in an intensive care unit for a long time on antibiotics, they can become deficient in Vitamin K, which is a factor they need for blood clotting. And that's probably an extreme example. 
- Do you know if someone has been on broad spectrum antibiotics for a while, what the number of gut microbes goes down to? 
- Only a guess at numbers. 
- I don't know that, I'm sorry. 

- Just asking for a hunch. You mentioned earlier that microbes stimulate our immune system. There are autoimmune diseases that are very mysterious, for example. 
- Right. 
- Some people have hypothesized that microbes that we can't attack could be causing some of those. 
- I think that's probably very likely to be the case. I would guess there are an enormous number of human diseases, or just, you know, human conditions that will turn out to be related to your microbial flora, that we don't even understand yet. Some are starting to come to the fore and starting to be understood. Heart disease in particular is very much linked to microbes. I'm sure autoimmune diseases, as well. 

I mean, in terms of a hospital setting, I read recently that, and I didn't know this before, people who are on IV feeding, now it turns out it is much better for the patient if you feed the nutrients into the gut first, not directly into the bloodstream, presumably because you need to keep your microbes fed also. Yeah, there are lots of things like that. 
- I was going to give the example of the c. difficile. If you've heard about that, people get an antibiotic and all of a sudden, you have a c. diff. outbreak. It's in your gut. It's kept down with the other flora. You lose that. 

The c. diff. is pretty resistant and it takes over, and you get very miserable. 
- Go ahead. 
- I suppose eukaryotic cells are often larger than microbes. So what fraction of their mass? 
- That's a good point. Your mass is probably mostly your own. 
(laughter)
 But you probably do have, I'm guessing a little bit, but I'd say, you know, ten pounds, at least, of microbes. 

Yeah? 
- Are there any analogies with what you're working with and involvement with chromosomes? 
- Absolutely. We do not study the bacterial cytoskeleton, but there is one. The bacteria have proteins which are evolutionarily related to all of those cytoskeletal and intermediate filament proteins that eukaryotic cells also have. I think that the motor mechanism maybe something like myacin, which is a motor protein in eukaryotic cells also. Although it's not really related closely at the sequence level, except right around the ATP binding site. Yeah? 
- You said that the retraction of pili was a single continuous process. Is that true also of the extension of pili? 

- Yes, for the most part. Again, that's not the sort of thing that has been measured, except by looking at micrographs of pili being assembled and dissembled. So it's a little anecdotal, but yes. Those retraction events, their force has been measured using a laser tweezer experiments. That motor is the strongest biological motor that we know about. And what is really cool is, those motor retraction events are additive. So if ten pili are all pulling on the same target at the same time, you get ten times the force. It's sort of like, you know, hooking up ten locomotives in a row. So you can really get. There are signaling pathways in human cells that are turned on by the retraction event alone. 

And there is a lab that has been able to take away the pili altogether, substitute it with magnetic force, and turn on the same signaling pathways in the eukaryotic cells. So this mechanical force is a real signal to the eukaryotic cells. And there's probably a lot more that we'll learn as time goes on about just mechanical force as a signaling mechanism. Yeah? 
- What's at the end of the pili? Are there special attachment structures that are created at the end? And if so, what about the extension? 
- You're on the forefront of microbiology with your question. About a decade ago, a paper came out, a beautiful electromicrograph with this tip protein labeled with an antibody. And everybody said, ah-ha, that's it. 

There's a tip protein. That's why it adheres specifically. And no one has ever been able to reproduce that result, even though it was published in a very high profile journal. And so, the answer is many people assume there is a tip protein. But it hasn't been demonstrated yet. A paper came out two days ago showing a potential tip complex. So you'll just have to stay tuned. Yeah, maybe. Yeah? 
- Is there any logic between the bacterial flagella and others, or the eukaryotic? 

- No, not so much at a protein sequence level. Maybe at a functional level. But to a certain extent, you know, a lot of these filaments are alpha helices that pack against each other. So, you know, at a structural level, there may be some overlap. But the bacterial flagella are a separate protein from the eukaryotic flagella. Yeah? 
- Tell us about laser tweezers. 
- Yeah, you should bring in someone for a laser tweezer night. That would be cool. Laser tweezers are essentially, and I've never done these experiments, so you have to really ask the experts. 

But essentially, you trap a bead in a laser beam. And then you can attach the tip of the pilis to a substrate. And you can measure the force that it takes to prevent the filament from being drawn back into the cell. So you can actually the displacement. And you use that as a readout of the force that is being generated in the first place. You can do this on tiny scales of individual molecules. Yeah? 
- There is another phenomenon in higher organisms of human cells, which is that there are receptors that move to the surface and depart from the surface under different signaling. 
- Yes. 
- So, do you know of homology of those mechanisms with what you're studying in bacteria? 

- So you mean if the filament would sort of be trafficked to the surface to do retraction and being taken away again? 
- As the protein emerges. 
- Is it always there? 
- It is dissembled or resynthesized? 
- It's interesting, because the secretion pathway actually does have some real functional homologies to this assembly and retraction pathway. But even more, the pathway, just for secretion of soluble proteins into the exterior of the bacteria, the crossing of the membrane has a lot of homologies to protein secretion in eukaryotic cells, right down to the structures of the proteins that are involved. Yeah, absolutely. 
- Does there appear to be any control over how long a pili is created? 
- No. The control over how long a pilis is, that was the question. 

How does the bacteria control how long the filament is. It seems to be regulated by the expression level of the proteins that form the base. So if you under express the protein that does the retraction, which means you would have correspondingly more assembly bases, you would get more pili. And you can also control the length of the pilis by under expressing the filament subunit itself. It's controlled at the level of how many proteins of each kind are made in a cell. Great. Thank you all very much. 
(applause)