Hello, I’m delighted to be here and I’m going to talk to you about my idea of tiny conspiracies which is how bacteria talk to each other. And my name’s Bonnie Bassler, I’m a professor in the biology department at Princeton University and I’m also a Howard Hughes Medical Institute investigator. The goals that I hope you come away with after this seminar is to understand that bacteria can communicate and then the deeper messages that I’d like you to get is that the process of bacteria communicating with one another is called quorum sensing and I’ll explain how that works, that what quorum sensing allows bacteria to do is to act like multicellular organisms and to take on tasks that they couldn’t accomplish if they were acting as individuals. They use multiple words to communicate, these words are going to be chemicals, and those words, I hope you’ll understand, allow bacteria to understand who is self and who is other, similar to what the cells in your body do, and then finally, I want to try to show you that there’s a practical application to this work, which is if we could either keep harmful bacteria from communicating, or help beneficial bacteria to communicate, those could be new kinds of therapeutics that could be developed in the future. So, to get going, I want to remind you all what a bacterium is. And so this is a picture of sort of a typical bacterial cell that would be like an E. coli, that’s found in your gut, and bacteria are the oldest organisms on the earth. They’ve been here for 4 billion years and they all look pretty much like this cartoon. And what I mean by that is that they’re single cells and they’re covered with a membrane and that keeps the outside world out and the inside world in. And then inside is the blue part, that’s the cytoplasm, and so that has all the biomolecules, like inside your cells, that keep the bacteria alive. What’s special about bacteria is that they all only have one piece of DNA, so this chromosome that’s running through the cell. So, they have very few genes, so only a couple thousand genes on this one chromosome, and that encodes all of the information that gives them all their parts that make them live and all their physical features as well as their behaviors. So, scientists, ok, scientists have known about bacteria for about 400 years, and we’ve been able to look at them under microscopes. And if you watch bacteria, what scientists have seen is that they consume nutrients from their environment, they grow to twice their size, they replicate their DNA, and put one in each end, one chromosome in each end, and then they cut themselves down the middle and one cell becomes two, and so on and so on. And so it’s kind of a boring, mundane life. They eat and they divide and they eat and they divide. And so for most of these 400 years, because it’s seemingly so simple, people thought that bacteria didn’t have very sophisticated behaviors. And what I mean by that is when the cell divides in half, each sister cell has no knowledge, if you will, of its sibling. So, they went out and did their own thing, as individuals. And they didn’t have behaviors that were more sophisticated that allowed them to know that one another were present. And so I want to try to convince you today that in fact they do understand their sisters and brothers, and their cousins and their enemies are around and they carry out group behaviors. But before I get into that, I want to just tell you a little bit of something about your relationship with bacteria. So, what I’ve done is to make my version of a man. This is a human being, and each of the circles inside the man are supposed to represent some number of the cells that it takes to make a human being. And we know there’s a few trillion cells, so that’s how many cells make your body and give you all of your features and all of your behaviors. So, you’re a few trillion cells. But it turns out that every moment of your life, you have ten times more bacterial cells in you, mostly in your gut, or on you, covering you in a biofilm, so you’re ten times more bacterial cells in you and on you, than human cells. So, you’re always associated with bacteria during your life. And of course, this is the 21st century, so we know a lot more about organisms than just cells and so now I’ve remade my man out of the A’s, T’s, G’s and C’s that make up the nucleotide codes of the famous double helix that make up your genetic information. And of course, we’ve sequenced the human genome, we know there’s about twenty or thirty thousand genes, so that’s how many bits of information it takes to make a human. It turns out that you have a hundred times more bacterial genes than human genes in you or on you every moment of your life. So, you can pick which of these two ideas you like. At best, you’re 10 percent human, probably you’re really only 1 percent human. The rest of you is bacterial. So, all your life is spent with these bacteria and these bacteria are beneficial to you. As I told you already, they cover you in a biofilm that keeps the environment from hurting you and they also supply you with all kinds of genes and proteins that you don’t have in your own genome. They help you digest your food, they make your vitamins, they educate your immune system. So, you inherit these bacteria from your parents, right after you’re born, and you keep them your whole life. And so what we understand now that we didn’t used to understand is that there would be no life on earth without bacteria. No human, no animal, no plant, no insect. So, they’re essential for keeping us alive. But of course, if you’ve heard about bacteria, you’ve probably heard something terrible, because we do know that bacteria, some bacteria, are also extremely harmful and can actually kill animals or plants or human beings. And so those are on the top, these are just a few notorious bacteria that you might have heard about that actually have no business being in you or on you. And when they do get in you or on you, they can make you really sick or they can kill you. And so bacteria have these two personalities. The bad ones that we hear about all the time and then these beneficial bacteria that are these invisible partners with us that keep us alive. And the question that my lab wanted to ask is that whether you think about these bad behaviors that bacteria do, or the good behaviors that bacteria do, we wondered, how do bacteria ever do anything at all? They’re so tiny, right, you’ve never seen one, you need a microscope to see a bacterium, and so if they’re really living as these tiny individuals, how can it be that they can have such a huge impact on the world. And so what I want to try to tell you is that bacteria act in groups and that allows them to carry out these magnificent tasks that they’re just too small to do on their own. And so to understand this idea that bacteria work as these multicellular groups, it actually came from a funny finding. There was a scientist named Woody Hastings who about 50 years ago was really interested in bioluminescent bacteria that lived in the ocean and the one that was his favorite was a bacterium named Vibrio fischeri that lives in a symbiotic association with this animal, which is called the Hawaiian bobtail squid. And we went to my friend Margaret McFall-Ngai’s lab to make this movie. So, now I’ll show you what the squid looks like. So, this squid is nocturnal. So, during the day, it likes to be asleep. And so what we’ve done is to put this squid in this aquarium and in the light. And it looks kind of spastic and disorganized because it hates being outside during the day. So, if we give that same squid some sediment, so just some pebbles, this is what the squid does. It likes to be awake at night, and asleep at the day. So, we’ve given it sediments and now it’s going to go to sleep. But it wants to stay away from predators, it doesn’t want predators to see it during the day, so it buries itself in the sediments, and then sleeps. So, we’ll just watch it. And what you can see is it’s very thorough, it is very serious about being completely hidden from predators because that’s an amazingly important feat if the squid wants to stay alive. So, now he’ll finish covering himself up, he retracts those tentacles, and if I showed you this aquarium, you wouldn’t know that there’s a squid in there. And of course, neither would a predator, because it’s invisible. So, this is a fantastic strategy during the day for the squid to be invisible. The problem is that at night, the squid has to come out to hunt. And so, when it comes out at night, since it lives just in sort of shallow waters, like in this aquarium, on bright nights when there’s lots of starlight or moonlight, that light could go through the water, and hit the squid’s back and cast a shadow. And so this is where the bacteria and this Vibrio fischeri symbiosis comes in. So now what you’re looking at is the squid, it’s been turned on its back, and I hope what you can see is in the middle, there’s a little glowing orb. This is a specialized light organ that housing the Vibrio fischeri bacteria. So, what the squid does is it has the bacteria in there at something like 10 to the twelfth cells per milliliter, packed, like paste, inside this squid, and those bacteria, this Vibrio fischeri are bioluminescent, so they make light. And so what the squid does is when it comes out at night to swim, it’s got this light organ that’s jam packed filled with this bioluminescent bacteria, so Vibrio fischeri is making light. And what the squid has, it has detectors on its back, so it can sense how much starlight or moonlight is hitting its back and then it opens and closes a shutter so it modulates how much light comes out of the bottom to exactly match how much light hits the squid’s back from the stars and the moon. And so, that way, by using the light from the bacteria to match the moonlight, the squid can counter illuminate itself and not make a shadow. So, it actually uses the light made by the bacteria in order, as an anti-predation device to keep itself invisible from predators that would see its shadow, calculate its trajectory and eat it. So, this is like the stealth bomber of the ocean, the squid cloaks itself in this invisible device and that keeps predators from finding it. So, this symbiosis helps the squid stay alive and the reason the bacteria provide the light, the evolutionary selection, is that the squid feeds them in this light organ, so they can make many more of themselves by living in this nutrient rich environment of the squid. But, now I want to change to the bacterial perspective. It turns out that the bacteria only make light at particular times. So, only when there’s lots of them together in this light organ. And that is controlled by this cell-cell communication mechanism that I told you about. So, now if we think about this from the bacterium’s point of view, it’s, remember, bacteria are tiny, so even in the squid light organ, they’re very small. And so what happens is when the bacteria are at low cell density, so when they’re basically alone, Vibrio fischeri, it turns out, makes no light under that condition. But what the bacteria do is that they make and release a small molecule that you can think of like a hormone, and we call it an autoinducer and that’s the red triangles on this slide. So, when the bacteria are at low cell density, so when they’re alone, they make and release this molecule, but the bacteria live in a big world. So, the molecule diffuses away. That tells Vibrio fischeri you’re by yourself, you should carry out individual behaviors. And in this case, that means, don’t make light. But when the cells grow and multiply, since all the bacteria in the environment are making and producing this molecule, right, they’re all contributing a share of the autoinducer, this molecule, the red triangles, increases exactly in proportion to cell number and when the molecule hits a particular threshold level, the bacteria detect it and all the bacteria turn on light in unison. So, the way the bacteria tell whether there’s enough of them together to make perceivable light is by measuring the concentration of this chemical, the autoinducer. And they’re using that as a proxy for cell number. So, only when they’re jam packed in the light organ of the squid, not when they’re free living in the ocean, will the bacteria make light, because that’s the only place that the autoinducer can accumulate to a significant concentration. So, this is how Vibrio fischeri talks to one another, with these chemical words, to tell the cells that there are neighbors around. So, the tools of molecular biology were brought to this to understand how could this work? And so what was found was that there was a gene and an enzyme that makes a protein, excuse me, there was gene, that makes a protein, an enzyme, that makes the autoinducer molecule, right, the red triangle. And that’s freely diffusible in and out of the cell, so the more cells there are, the more of that autoinducer there is. And then there’s a partner protein, a receptor, that sits on the bacterial cell membrane that detects that autoinducer, and so when that autoinducer increases to a particular threshold level, it can find that receptor, lock in there, and that sends a signal into the cells to tell Vibrio fischeri to turn on the genes that make the enzymes that make light. And so this is a very simple circuit; the more cells there are, the more of the autoinducer there is. At a particular concentration, which means something about cell number, all the bacteria turn on light in unison. And so this was originally described in Vibrio fischeri, but in the past 20 or so years, scientists have been finding these communication circuits in all kinds of diverse bacteria. So, now there are hundreds and hundreds of examples of different species of bacteria that all have an enzyme that makes an autoinducer, there’s always a partner receptor, and then together, these information circuits tell bacteria to turn on all kinds of genes that they want to express when they’re in a community, but not when they’re alone. And so now we have a name for this process, we call it quorum sensing. The bacteria vote with these chemical votes, the vote gets tallied, and all the cells go along with the votes. And so what you can see is this allows bacteria to act as multicellular organisms and to carry out collective behaviors. And so I told you about bioluminescence in Vibrio fischeri but what scientists have gone on to learn is that each species of bacteria typically has hundreds of genes, hundreds of behaviors, that they want to carry out as a group. And so among those behaviors, besides bioluminesnce, are things like biofilm formation, so how bacteria sit on surfaces, and coat surfaces and also the group or collective expression of virulence factors. So, what we now know is that when pathogens, when harmful bacteria, get into the body, they can’t make an infection by themselves, it’s only when they act together to release their toxins or their poisons, that these infections become successful. So, now we understand that if you make pathogenic bacteria so they can’t talk, or they can’t hear, those bacteria aren’t pathogens. And so I hope what you can see is that all of these behaviors are when a small bacterium is encountering a big world and trying to change it. So, these kinds of behaviors need lots of cells working together, and then the behaviors, whether it’s symbiosis in Vibrio fischeri, or pathogenesis, those behaviors become successful. So, that’s how the process works and a lot chemistry has also been done to try to understand well, what are these words, these chemical words, that the bacteria are talking with. And so the first word that was discovered was Vibrio fischeri’s because that’s the bacterium that’s been studied the longest. So, on my cartoons, I had shown you these red triangles. But these are really molecules. So, this is the Vibrio fischeri molecule that it uses to communicate. And then scientists started looking at other species of bacteria and what I put on this slide is just a smattering of different species of bacteria and the word that each one uses to communicate. And what I hope you can see is that on the left side of all of the words, the chemical is identical. But the right side, these chains, these are just carbons, those are a little bit different in each species of bacteria. And what that does, those small changes, is to confer exquisite species specificities to each of these languages. So, each of these molecules locks, really like a lock and key, into its own receptor and no other. So, what I mean by that is if I take the Agrobacterium molecule and put it on Vibrio fischeri, nothing happens, And likewise, the Vibrio fischeri molecule has no effect on Agrobacterium. So, these words enable intraspeices communication. This is how bacteria talk to their brothers and sisters, they have private, secret conversations to understand when there’s enough of their siblings around that they should all take on some group task. So, once we got this far in our studies, we started to understand that bacteria can talk and they have these collective behaviors, like symbiosis, like pathogenesis, but then we started to wonder, well, wait a minute, most bacteria don’t get this wonderful life like Vibrio fischeri has. I mean, Vibrio fischeri gets to live in a one to one relationship with this squid. Most bacteria live more like this. What you’re looking at right now, this is human skin, and this is a biofilm on your skin. And so, as I already told you, you’re covered in bacteria and this shows what your skin would look like under the microscope and what I hope you can see is all these different shapes and sizes. Every one of those different shapes and sizes of bacteria is a different species. And so what we thought is that if bacteria are really trying to count their numbers and these languages are so species specific, what do they do when they find themselves in this kind of situation. How do they count? And so we thought we could take advantage of this bioluminescence business again in order to study this. So, I told you a story about Vibrio fischeri. We also knew about a cousin of Vibrio fischeri’s, a bacterium named Vibrio harveyi. This is a bioluminescent marine bacterium that lives in the ocean but it lives free-living. And so we thought, well, this guy doesn’t get this nice pristine life with this squid. This guy has to encounter a changing environment, and all kinds of other bacteria. Maybe if we study Vibrio harveyi’s quorum sensing circuit, we would find out something more sophisticated about how a free-living bacterium works. And so what I did on this slide is to just show you what my lab does. What you’re looking at, this is a flask of a high cell density culture of Vibrio fischeri, here’s some Petri plates, this is my guys goofing around, writing my name with Q-tips, in bioluminescent bacteria. And to take this picture, all we had to do was turn the lights off in the room and this is what we see. So, this picture was taken from the light that the bacteria are making. And so you’ll remember that my lab is interested in how bacteria talk to each other. Right, but you can’t see bacteria, so you can’t see whether they’re talking or not talking. What’s so fantastic about bioluminescence and why I think this whole field got discovered in bioluminescence is because you can just turn the lights off and bioluminescence makes this invisible world of bacteria visible. So, we’re geneticists, what we can do is make mutants and then just plate them out on these Petri plates and look for Vibrio harveyi that aren’t making light when they should be or are making light when they shouldn’t be. And just by doing this simple thing of looking on the Petri plates for dark and bright cells, we could find the components that make up the Vibrio harveyi quorum sensing circuit. So, that’s what we did, so we made mutants that were goofy for bioluminescence, we took advantage of this natural read-out of light and what we found was that of course, sure enough, Vibrio harveyi had an intraspecies communication system, just like Vibrio fischeri’s. I told you that we think all bacteria probably have that but what we also found in Vibrio harveyi was a second system. When we looked further in our mutants, we found that there was a second enzyme, that made another molecule and it had a partner receptor. And so now we see that there are two languages, and all of this information comes in to tell Vibrio harveyi to turn on and off light and hundreds of other quorum sensing behaviors, but remember, we’re only measuring bioluminescence in our experiment. And so we thought, well, that’s interesting, there’s this intraspecies system that’s very specific, what’s this second system and what we did was to purify this second molecule and we found that all bacteria made this molecule. So, we thought, that means that this is the interspecies communication system. This is how bacteria talk across species. And so now I’ll show you the molecule. This is the molecule that we call autoinducer 2 because it’s the second one we found and it’s one thing. It’s a five carbon molecule that’s shown on this slide and in my former slide, it was the pink ovals, as a cartoon, but what you need to understand is that unlike the intraspecies communication molecules which are each a little bit different from the other, in this case, every bacteria makes exactly the same molecule. They make this. So, the second molecule, the universal language, is one molecule, and that’s why bacteria can talk between species, it’s because they use the same word. So, now what we think is that probably all bacteria are at least built like this, in that they’re bilingual, or multilingual, in that they have some way to know self, so they have an intraspecies communication system, and then they use autoinducer 2 and this partner receptor to know other. And so the computation that we think bacteria do is the following. We think when bacteria find themself in some interesting situation, the first thing they do is they just scan the environment, using these receptors, for molecules. And they say, am I alone, or am I in a group? And that starts to tell them what genes to turn on and off. Individual or group behavior genes. And then the more sophisticated computation they do is that they measure the ratio of these two molecules, and by doing that, they can ask is my species in the majority, and the other species in the minority, or the reverse? And then they change the genes that they turn on and off which changes the behaviors they carry out based on who’s winning and who’s losing in any given mixture. And so now we understand that these bacteria, probably all bacteria, communicate, they carry out group behaviors, there’s probably many more molecules to find, but a practical part of this work that we’re thinking about is what I’ve alluded to, is that we know that both good bacteria and harmful bacteria use quorum sensing and we know that these group behaviors require quorum sensing to function. So, for example, pathogens, if you make mutants in quorum sensing they can’t turn on virulence. So, what we’re wondering is can we deliberately interfere with these quorum sensing cascades in order to make new kinds of therapeutics. And there’re sort of two strategies, one would be if you want a very species-specific therapeutic, like a drug for a particular bacteria, you would like to go and target this intraspecies communication system. In other cases, perhaps you get sick and your physician doesn’t know what’s wrong with you, maybe you’d like to be treated with a broad spectrum therapeutic. In that case, perhaps we’d like to take advantage of this universal, or interspecies communication circuit. So, we’ve made molecules that interfere with each of these kinds of circuits and they actually shut down quorum sensing in a test tube. And so the experiment to see whether these could in therapeutics is to use an animal model. So, the experiment that we’re doing is that we have pathogenic bacteria that can kill an animal host. Right, so if you give an infection to the animal, the animal will die. And so now what we can take are our anti-quorum sensing molecules, and so I’ve made them this funny shape, these are molecules that block the quorum sensing receptor, and when we give those along with the pathogenic bacteria, the pathogenic bacteria can no longer do quorum sensing, and so what happens is that in fact, the animal lives. And so these are not therapeutics yet, but it’s very exciting for us because it tells us that there’s merit to this idea of instead of killing bacteria, the way normal antibiotics do, if we could just alter their behavior, perhaps we could think of new ways to treat bacteria and hopefully you know that there’s a global problem with not enough antibiotics, or new antibiotics, to fight off all the pathogenic bacteria that people and agriculture are dealing with. And so we’re exploring this now in the hopes that we could think of new ways to battle bad bacteria. But I also want to just say, even though I’m on this cartoon, is to just remind you of the beginning of my talk, which is that most bacteria are really good, they keep us alive, they keep plants alive, they keep animals alive, so beyond making anti-quorum sensing molecules to interfere with pathogenic bacteria, we’re also trying to make pro-quorum sensing molecules, so maybe if we could learn enough about the good bacteria that live in us or on us, we could enhance their communication circuits in order for them to do things that are beneficial for humans or for agriculture or for industry. And so those are sort of the practical areas that we’re going to based on the sort of basic science that we learned with these bioluminescent bacteria. And so, I want to go through what I think the high points of this talk were. I hope what you think now is that bacteria can talk to each other. They don’t use words like we use, they use a chemical language. And this process, which allows them to carry out collective behaviors, is called quorum sensing. Beyond talking to each other, what quorum sensing allows bacteria to do is to synchronize their behavior so they can act as a group. And of course, I’ve told you that bacteria are the oldest living organisms on the earth and so we believe that the principles that underlie these group behaviors in bacteria are interesting for scientists to think about so that we can understand how multicellularity developed in higher organisms. We think that many of the features that are encoded in these quorum sensing cascades will be preserved in higher organisms. And of course, I’ve also told you that there’s at least two molecules involved in this process, one that tells bacteria, “me” and one that says “other’ and so we think that that enables bacteria to be sophisticated enough to tell self from other and again this is what the cells in your body do. Your kidney and your heart cells don’t get all mixed up because they each have their own sets of chemicals to tell cells, different groups of cells, to do different tasks. Again, we think that that was invented by the bacterial world. And then finally, there’s a practical part of this work, which is if we could impede quorum sensing by making molecules that stop bacteria from talking to each other, we could treat harmful bacteria, and likewise, we can hope we can improve quorum sensing in beneficial bacteria or industrially important bacteria in order to make new industrial and medical therapies. And then I want to finish by showing you my lab, I’ll get out of the way. This is my gang at Princeton University, I’m really proud of them, they made all of the discoveries that I told you about in this talk and then I just want to show you that I hope, as you’re watching this, that they look a lot like you, these are college kids and graduate students, right, and they’re just regular people that think science is cool and mysterious and magical and they find this fun and I find working with them fun and I hope that if you’re interested in science at all, that you’ll give research a try, because it’s really a blast to do it and these are the kind of people you’ll be doing it with, they’re just a hoot. And so thank you for listening to me, and good luck.