Cell-Cell Communication in Bacteria via Quorum Sensing
Transcript of Part 1: Bacterial Communication via Quorum Sensing
00:00:03.14 Hi, my name's Bonnie Bassler and I'm a professor at Princeton University 00:00:07.01 and I'm also an investigator at the Howard Hughes Medical Institute 00:00:10.17 and I'm delighted to be here to get to talk to you about bacterial quorum sensing. 00:00:15.00 So the goal of my two seminars is to try to convince you that bacteria can talk to each other 00:00:21.10 and to try to show you that what this chemical communication does 00:00:24.29 is to allow bacteria to act as enormous multicellular organisms and 00:00:30.12 accomplish tasks that they could never accomplish if 00:00:33.13 they simply acted as individuals, because they're too tiny. 00:00:37.07 And so what I thought I would do today is to give you sort of a historical tour 00:00:42.10 of how we got into this idea that bacteria can talk with each other 00:00:45.13 and then try to give you some ideas of where this field is going 00:00:48.18 and how we're trying to do something that's medically relevant 00:00:51.22 by interfering with bacterial communication. 00:00:54.01 So, to get going, the idea that bacteria can talk to each other all started 00:00:59.21 about 40 years ago when a man named Woody Hastings made an amazing observation. 00:01:04.00 So what he noticed was that there were two bioluminescent bacteria, 00:01:08.15 so bacteria from the ocean that made light, sort of like firefly light, 00:01:13.06 but what he noticed was a special property of the light. 00:01:16.05 That they only made light when they were at high cell density. 00:01:18.29 And so this is actually depicted on my first slide. 00:01:21.15 What you're looking at is a flask. 00:01:23.13 This is just a person from my lab holding a flask of a liquid culture of one of these two bacteria. 00:01:29.08 They're called Vibrio fischeri and Vibrio harveyi. 00:01:32.20 And what you can see is that the flask...or the liquid in the flask is glowing in the dark. 00:01:37.22 And that light is made by the bacteria, in this case, Vibrio harveyi. 00:01:42.04 And what Woody Hastings noticed was that at low cell density, 00:01:45.27 so when the bacteria were alone, they didn't make light. 00:01:48.29 But, when they grew to a certain cell number, all of the bacteria turned on light together. 00:01:53.29 And so the question that he wanted to know, and what we're going to talk about today is: 00:01:57.21 "How can it be that a bacterium knows the difference between times when it's alone 00:02:02.20 and times when it's in high number?" 00:02:05.04 And what's fantastic about bioluminescence is that you can just see it. 00:02:09.14 So, we're geneticist in my lab and we want to understand how these bacteria 00:02:13.16 perceive when they're alone and when they're together. 00:02:15.22 And so what we can do is to just to make mutants, turn the lights off, 00:02:19.27 look at the Petri plates and look for bacteria that are glowing when they shouldn't be 00:02:24.01 or not glowing when they should be. 00:02:25.22 And just by doing that simple experiment, we and many other groups in the field 00:02:29.26 have been able to understand how these bacteria tell the difference between 00:02:33.26 times when they're alone and times when they're together 00:02:36.12 and then initiate a signal transduction cascade, 00:02:39.13 the output of which is this beautiful bioluminescence. 00:02:42.23 So, I told you that Woody Hastings noticed this both in two bacteria, 00:02:46.23 one was named Vibrio harveyi, which is this free living bacteria that you're looking on at this slide 00:02:51.14 and the other one was its very close relative Vibrio fischeri. 00:02:55.07 So like Vibrio harveyi, Vibrio fischeri is a bioluminescent marine bacterium. 00:03:00.16 But it lives a very different lifestyle than Vibrio harveyi. Vibrio harveyi's free-living 00:03:05.06 but Vibrio fischeri lives as a symbiont in a number of marine animals 00:03:09.15 one of which is this one, which is called the Hawaiian bobtailed squid. 00:03:12.26 So what you're looking at is a squid that's been turned on its back 00:03:16.13 and dissected down the middle and I hope that you can see are these two glowing lobes 00:03:21.23 that make up a specialized light organ in the squid 00:03:24.16 and this is the place that the bacteria live. 00:03:26.29 So the way this symbiosis works is: the squid is, 00:03:29.29 this light organ is under the bottom or the mantel of the squid. 00:03:32.24 This light organ is under there and it houses Vibrio fischeri 00:03:36.06 at something like ten to the eleventh or ten to the twelfth cells per mL. 00:03:40.07 We have no idea how the squid grows the bacteria to this high cell number. 00:03:45.00 But what we know is that the bacteria are trapped inside the light organ 00:03:48.26 and in there they make a molecule that you can think of like a hormone 00:03:53.04 or a pheromone and we call it an auto-inducer. 00:03:55.25 So they make and they release this molecule 00:03:58.12 and since the bacteria are trapped inside the light organ of this squid 00:04:02.16 the molecule gets trapped inside with them 00:04:05.06 and it tells the bacteria "You're inside, not outside. So, you should make light." 00:04:09.28 And so Vibrio fischeri glows because it perceives this molecule 00:04:13.15 which helps it count its neighbors. 00:04:16.20 And so the reason Vibrio fischeri does that is because this squid feeds it. 00:04:20.17 So this light organ is loaded with amino acids and nutrients and all kinds of goodies 00:04:25.10 that makes the life of the bacterium much easier than 00:04:28.10 if the bacterium was living free-living in the ocean. 00:04:31.04 So the selection from the bacterium's point of view is that it gets fed 00:04:34.23 and gets to grow to high numbers in that squid. 00:04:37.02 Now the reason the squid is interested in this is because it wants the light from that bacteria. 00:04:41.18 So the way this work from the squid's perspective is: 00:04:44.13 this little squid, its only about this big full grown 00:04:47.05 and it lives off the coast of Hawaii, that's why its called the Hawaiian bobtailed squid 00:04:51.07 and the squid is nocturnal. So, during the day it buries itself in the sand and it sleeps 00:04:56.22 but then at night it has to come out to hunt. 00:04:58.27 And so on bright nights when there's lots of starlight or moonlight, 00:05:02.16 since this squid lives just off the coast, so just this sort of shallow, knee-deep water 00:05:06.22 since its living in this shallow water, the light from the moon and the stars 00:05:10.20 can penetrate the water that the squid lives in. 00:05:12.23 And so what the squid has developed is a shutter. 00:05:15.12 It's the squid's ink sack that it can open and close over this specialized light organ. 00:05:20.17 And so what happens is that the squid has detectors on its back 00:05:24.00 so it can sense how much moonlight and starlight is hitting its back 00:05:27.07 and then it opens and closes this shutter so the amount of light 00:05:30.15 coming out of the bottom, which is made by the bacteria, 00:05:32.24 exactly matches how much light hits the squid's back. 00:05:36.05 So this way the squid doesn't make a shadow. 00:05:38.16 So it actually uses the light from the bacteria to counter-illuminate itself 00:05:42.29 as an anti-predation device. 00:05:44.05 And so the selection for the light from the squid's point of view is that 00:05:48.00 it gets protected, for its lifetime, from predators. 00:05:50.17 So the bacteria make the light, they get fed and the squid uses the light to protect itself. 00:05:56.09 But of course this squid has this terrible problem 00:05:58.19 because it's got this dying, ten to the twelfth cells per mL culture of bacteria 00:06:02.18 in this little light organ and it can't maintain that. 00:06:04.29 And so what happens is every morning, when the squid goes back to sleep, 00:06:08.24 when it buries itself in the sand, its got a pump that's attached to its circadian rhythm. 00:06:13.02 And so when the sun comes up, what the squid does is it pumps out 95% of the bacteria. 00:06:18.26 And so now the bacteria are dilute and of course that little molecule, 00:06:22.04 that hormone, auto-inducer molecule I told you about, is gone. 00:06:25.18 And so now the bacteria can't perceive that molecule, so they don't make light. 00:06:29.12 But then as the day go by, the bacteria start doubling, they keep releasing this hormone molecule 00:06:34.18 and at night, they perceive it, and the light comes on exactly when the squid needs it. 00:06:39.13 And so the reason to tell you about these two, first stories is because 00:06:43.09 now we understand that hundreds of species of bacteria make auto-inducers 00:06:47.11 and talk to one another with these chemical communication systems. 00:06:52.22 So, the way we understand this in the simplest terms is that at low cell density, 00:06:57.25 so this is over here, when a bacterium is alone or in dilute suspension, 00:07:01.21 it's making and releasing these molecules that I have as these little blue dots. 00:07:05.16 But, of course, if the bacteria is dilute, the molecules just diffuse away. 00:07:09.13 But as the cells grow in number, when the population density increases, 00:07:13.19 since all the bacteria are releasing these molecules into the environment 00:07:17.05 the more cells there are, the more of this molecule there is. 00:07:20.12 And at a particular concentration of the molecule, which is indicative of cell number, 00:07:25.02 the bacteria perceive that the molecule is there 00:07:27.16 and then they all change their gene expression in unison. 00:07:31.03 And so in the case of Vibrio fischeri and Vibrio harveyi, they turn on light. 00:07:35.08 And so what you can see then is that these bacteria are acting like an enormous 00:07:39.23 multicellular group and turning on and off gene expression, 00:07:43.04 which is really changes in behaviors, on a population wide scale. 00:07:47.00 And so now we have a fancy name for this: we call it quorum sensing. 00:07:50.22 The bacteria vote with these little chemical votes, they count the votes, 00:07:54.20 and then everyone responds to the vote. 00:07:57.08 And what I hope you'll think by the end of this seminar is that this had to be 00:08:00.28 one of the first steps in the development of multicellular organisms. 00:08:05.29 So this is sort of how it works in generic terms and we also know a lot now. 00:08:10.20 In these past few years we've learned a lot about the mechanisms 00:08:14.13 that underlie these communication systems. 00:08:16.16 And so there's a couple of sort of general themes that are emerging. 00:08:20.29 And so what we now understand is that in gram negative bacteria, 00:08:24.23 they typically have systems like the one I have depicted on this slide. 00:08:29.00 So this...these ovals will be my cells 00:08:33.23 these are supposed to mean bacterial cells from here forward. 00:08:36.14 So what we know about gram-negative bacteria, 00:08:38.22 is that they have an enzyme which is called LuxI, for the inducer. 00:08:43.17 And this is the enzyme that makes the signal molecule, which on this slide are these red diamonds. 00:08:48.25 And all of these signal molecules are what are called acyl homoserine lactones. 00:08:52.24 And I'll tell you more about those on the next slide. 00:08:54.26 So what we now understand is that hundreds of species of gram-negative bacteria 00:08:59.09 all have highly conserved LuxI enzymes 00:09:02.07 that make an acyl homoserine lactone signal molecule 00:09:05.03 and that freely diffuses in and out of the cells 00:09:07.24 so the more cells there are the more the molecule there is. 00:09:10.16 And when the molecule hits a particular amount 00:09:12.25 it gets bound by a partner protein that's called LuxR for regulation. 00:09:17.13 And what happens is when LuxR binds the auto-inducer molecule 00:09:21.05 that unveils a DNA binding domain on the LuxR protein. 00:09:25.04 So this complex of the LuxR protein and the signal molecule bound 00:09:29.13 can sit at the promoters of genes that the bacteria want to express 00:09:33.23 when they're in a community and they turn them on. 00:09:36.17 And so what this allows bacteria to do is to count their numbers 00:09:40.07 and then turn on genes that are useful only when the bacteria are acting as a group, 00:09:45.06 but are not useful when the bacteria are acting as individuals. 00:09:48.24 So again to reiterate, we now know that there are hundreds of systems like this 00:09:52.20 in gram-negative bacteria. So that's how the systems work. 00:09:56.26 We also know a lot about these signals that are made by these LuxI-type enzymes. 00:10:01.16 So what I did on this slide was just put a few different species of bacteria, 00:10:05.24 these are gram-negative bacteria that have quorum sensing. 00:10:08.27 And then I have the molecule that they use to talk with next to them. 00:10:12.22 And so what I hope you can see is that all of the molecules are related. 00:10:16.23 So again these are called acyl homoserine lactones. 00:10:19.20 And so what you see is that the right hand part of all of the molecules is identical. 00:10:24.07 That's the acyl homoserine lactone or the homoserine lactone. 00:10:27.15 But this left part, see these carbon chains? 00:10:30.04 Each one is a little bit different in every single species of bacteria. 00:10:34.18 And so what that does is to confer exquisite species specificities to each of these languages. 00:10:40.20 So what I mean by that is that only the bacterium that makes the signal can respond to it 00:10:45.23 and no other bacteria can. So for example, 00:10:48.19 if we take the Pseudomonas signal and but it on Vibrio fischeri nothing happens. 00:10:52.26 And likewise, Pseudomonas is impervious to the Vibrio fischeri signal. 00:10:57.18 So these molecules fit like locks and keys with their partner LuxR proteins 00:11:02.24 and there is no cross-talk in these systems. 00:11:05.15 They are for intra-species communication 00:11:07.29 and so what we think is that this kind of quorum sensing 00:11:10.29 allows bacteria to count their siblings. 00:11:13.15 It allows them to count and talk within their own species. 00:11:17.11 So that's how gram-negative bacteria do it. 00:11:20.05 We also now know that gram-positive bacteria have quorum sensing. 00:11:23.23 And so the ideas are similar to what I've already told you 00:11:26.16 but the mechanism that gram-positive's use to communicate is a little bit different. 00:11:30.19 So here I have sort of a model for gram-positive bacterial quorum sensing. 00:11:35.18 So gram-positive bacteria use peptides as their words or as their signals. 00:11:40.28 And so the way that this works is that the peptides are encoded in genes 00:11:45.13 and they get made as a precursor protein. So sort of a big protein. 00:11:48.27 And then there's a processing system that cuts that big protein into a smaller peptide 00:11:53.14 and then that gets secreted outside of the cell by a dedicated secretion machinery. 00:11:58.04 But again, the idea is the same: 00:12:00.05 the more cells there are, the more of this peptide there is accumulating in the environment. 00:12:04.08 And in this case, when the peptide hits a critical amount, 00:12:07.08 it gets bound by a transmembrane bound protein 00:12:11.10 so sort of a sensor that connects the inside of the cell to the outside. 00:12:15.03 And when the peptide gets bound, that initiates a phosphorylation cascade. 00:12:19.18 The bottom line of which is that, ultimately, a transcription factor gets phosphorylated. 00:12:24.16 That activates it, it lets it sit on the DNA and turn on the genes that the bacteria want 00:12:29.25 to express when they're a community. 00:12:31.26 So even though the mechanism is different than in the gram-negative bacteria, 00:12:35.20 the idea is really the same. The number of auto-inducer molecules, peptides in this case 00:12:41.10 increases with cell number increasing, 00:12:43.22 and then information gets sent in to a DNA binding protein 00:12:47.15 that turns on all of the genes the bacteria need to carry out group behaviors. 00:12:52.15 So that's how these work in gram-positive bacteria. 00:12:55.02 And just like I told you for gram-negative bacteria, we also know about the peptide signals. 00:13:00.00 So again, these are just a couple of peptides that we know are auto-inducers 00:13:03.23 for different gram-positive bacteria. And they can be a little bit fancy. 00:13:09.04 So sometimes its just the naked peptide or occasionally they'll have moieties stuck on them. 00:13:13.23 That's what this asterisk is supposed to mean. 00:13:15.12 Or they can be cyclized. So they put a few bells and whistles on these signals. 00:13:18.18 But the bottom line is they're peptides and again, in every case, 00:13:22.26 each species of gram-positive bacterium has its own peptide. 00:13:27.07 So these are private languages that are species specific. 00:13:30.15 And so, gram-positive bacteria use this to count their own cell number for intra-species communication. 00:13:37.07 And so that's how these systems work. 00:13:39.10 And now I want to just tell you a few of the behaviors 00:13:41.19 that are controlled by quorum sensing in different species of bacteria. 00:13:45.13 So I already told you how it works for Vibrio fischeri, 00:13:48.08 this bioluminescent bacteria that lives inside this squid. 00:13:51.10 So that story you've already gotten. 00:13:53.21 There's another bacterium, Pseudomonas aeruginosa, 00:13:56.06 which is a terrible opportunistic human pathogen. 00:13:59.10 This bacterium lives in the soil and it's relatively harmless unless one has cystic fibrosis. 00:14:05.06 And so probably you know that people who have CF have a genetic mutation in their lungs 00:14:10.12 so that they can't clear or sterilize their lungs. 00:14:13.13 So you and I breathe in all kinds of bacteria but we have mechanisms to keep our lungs clean. 00:14:18.06 People who have cystic fibrosis can't do that. 00:14:21.06 And so what happens is they have an infection in their lungs 00:14:24.28 that has all kinds of different bacteria in it. 00:14:27.28 And for reasons that we don't really understand, typically a person who has CF 00:14:33.02 in his or her teens will become permanently colonized by Pseudomonas aeruginosa. 00:14:37.29 And this is the bacterium that kills them. 00:14:40.10 And the reason is because Pseudomonas has about a hundred genes 00:14:44.11 that are controlled by quorum sensing, all of which are important for virulence. 00:14:48.18 And so what happens is that Pseudomonas gets into the lungs 00:14:51.24 and it makes what's called a biofilm. That's how bacteria stick in us or on us. 00:14:56.26 So it sits down, it adheres to the lungs, and it secretes all kinds of terrible virulence factors: 00:15:02.03 proteases and hydrolases that damage the person's lung tissue. 00:15:06.29 And all of these genes are controlled by quorum sensing. 00:15:10.10 And so even though this is a terrible disease, 00:15:12.26 if you actually think about this from the bacterium's point of view, 00:15:16.01 it's a fabulous strategy for being a pathogen. 00:15:19.07 The last thing a bacterium wants to do is to get in and when it's at only a few cells 00:15:24.14 start secreting all of these toxins and virulence factors. 00:15:28.01 That's why your immune system evolved. 00:15:29.17 It evolved to do surveillance and get rid of pathogenic bacteria. 00:15:32.28 So the strategy that Pseudomonas uses 00:15:35.09 is to get in, to wait, and to count itself with these small auto-inducer molecules 00:15:40.16 and to recognize when it has the right number 00:15:43.13 that if all of the bacteria secrete these virulence factors 00:15:46.24 in unison they'll be able to infect an enormous host. 00:15:50.24 And so what I hope you can see is that even though the disease is devastating 00:15:54.08 from the bacterium's point of survival, it's a really good strategy 00:15:58.00 to wait until you know there's enough of your friends around, if I can say that, 00:16:02.16 that if you do this thing together, you will be successful. 00:16:05.22 And so that's basically how we understand quorum sensing. 00:16:08.21 Another couple of examples, Agrobacterium tumefaciens is a plant pathogen. 00:16:14.03 And this pathogen causes crown gall tumors on plants 00:16:17.03 and it's a problem in the agricultural industry. 00:16:19.04 And what it uses for...with quorum sensing is mating. 00:16:22.27 So Agrobacterium has the virulence genes on a mobile piece of DNA. 00:16:27.12 And so what happens is when some of them start infecting the plant 00:16:30.17 what they do in response to quorum sensing is to start transferring this plasmid around. 00:16:35.17 So they make the entire community more infective than it was originally. 00:16:40.04 And so again if you want to give your DNA to somebody 00:16:42.25 you want there to be a recipient there. So that should be a behavior that's social: 00:16:47.11 that lots of cells need to do it together. 00:16:49.17 And then finally, I just put one more on here. 00:16:51.23 This one is called Erwinia carotovora. This is a plant pathogen. 00:16:55.17 You've seen this one. It's the one that turns your lettuce 00:16:57.11 and your potatoes brown in your refrigerator. 00:16:59.13 And it's a lot like Pseudomonas in that what it does is it waits and it counts its cells 00:17:04.11 and it recognizes when there's lots of cells there 00:17:06.28 and then they secrete together all of their virulence factor to make 00:17:10.16 a wound in the plant. But then what it does, it also has this very insidious strategy, 00:17:15.28 because simultaneous to that, what Erwinia also does, is to secrete all kinds of antibiotics 00:17:22.04 that it has immunity to but that other competitor bacteria 00:17:25.27 in the environment won't be immune to. 00:17:28.13 So it keeps the wound for itself and its siblings 00:17:31.06 and it fights off all the other competitors by killing them with these antibiotics. 00:17:35.18 And so this list goes on and on and on. 00:17:38.07 And I hope that what you can sort of see from this list 00:17:40.21 is that all of these kinds of behaviors 00:17:42.28 are behaviors that cells need many, many cells 00:17:45.29 acting in synchrony to make the behavior effective. 00:17:49.23 And so that's what we understand about quorum sensing, 00:17:52.02 that this is the beginning of multicellularity and these bacteria are carrying out tasks 00:17:57.17 that they could never be successful at if they simply acted as individuals 00:18:03.02 because they're too small, individually, to have an impact on the environment. 00:18:07.15 So, if you remember from the beginning of my talk, I told you that Woody Hastings 00:18:12.03 discovered this business of these bacteria communicating with molecules 00:18:16.05 in two different luminescent bacteria. Vibrio fischeri, that I've told you about 00:18:20.05 and also its relative Vibrio harveyi. 00:18:22.14 And so you might recall that I told you that Vibrio harveyi is a bioluminescent marine bacterium. 00:18:27.09 It has quorum sensing, but it lives a different lifestyle than Vibrio fischeri. 00:18:31.05 It's a free-living bacteria. And so we were interested in understanding 00:18:34.11 "How does a free-living bacterium in the ocean achieve quorum sensing?" 00:18:38.16 And so we did what I told you already, 00:18:40.28 which is to simply make mutants in Vibrio harveyi, 00:18:43.28 put them on Petri plates and look for bacteria 00:18:46.23 that weren't making light under conditions they should be 00:18:50.06 or were making light under conditions that they shouldn't be. 00:18:53.08 And just by doing that very simple experiment we were able to find the genes 00:18:58.09 involved in the quorum sensing system for Vibrio harveyi. 00:19:01.05 And so this is the model we've come up with for Vibrio harveyi. 00:19:05.25 And this is supposed to be the inside of the bacterium. 00:19:09.00 This over here is the periplasmic space. 00:19:11.15 And then way out there would be the outside. 00:19:14.03 Right, so this is supposed to be the bacterial membrane. 00:19:17.09 And much to our surprise, when we started working on Vibrio harveyi, 00:19:21.20 what we noticed was that the bacterium had a completely different quorum sensing system 00:19:26.25 than Vibrio fischeri and all these other LuxIR bacteria 00:19:30.16 that I told you about in the beginning of the talk. 00:19:32.26 What we realized right away is that the bacteria made two different autoinducers. 00:19:37.26 And so we named them in the order we found them 00:19:40.01 Autoinducer-1 and Autoinducer-2. 00:19:42.21 Autoinducer-1 is an acyl homoserine lactone. 00:19:46.16 So it's one of these run of the mill quorum sensing autoinducers 00:19:48.28 that's typical of gram-negative bacteria. 00:19:51.07 And actually there's a picture of it on my last slide. 00:19:53.16 The second autoinducer, autoinducer-2, was clearly not a homoserine lactone. 00:19:58.29 So homoserine lactones have very standard biochemical properties. 00:20:02.14 They all sort of behave the same biochemically. 00:20:05.00 And it was very clear to us that autoinducer-2 was something different. 00:20:08.18 And I'm going to spend a lot of time telling you about autoinducer-2 as we go along. 00:20:12.11 Each of these autoinducers was detected by its own sensory system. 00:20:16.25 So autoinducer-1 was detected by a protein that we named LuxN 00:20:20.24 and two proteins called LuxP and LuxQ work together 00:20:24.17 to detect and send the information in from autoinducer-2. 00:20:28.03 So LuxN and LuxQ and all of the other colored proteins that I have in this slide 00:20:33.15 are what are called bacterial two-component signaling proteins. 00:20:36.27 And these are highly conserved proteins in all kinds of species of bacteria. 00:20:41.15 And they're all set up the way that I have on this slide, 00:20:44.15 which is that they have a transmembrane domain that connects the inside of the cell 00:20:49.02 to the outside of the environment. 00:20:50.26 And so these transmembrane domains are responsible for detecting 00:20:54.12 things that are happening on the outside. 00:20:56.22 In the case of today's talk what they're detecting are these autoinducers. 00:21:00.08 And then that information gets sent into the inside of the cell 00:21:03.27 by a phosphorylation cascade that always involves histidines and aspartates. 00:21:08.28 The way that this signaling system works is that at low cell density, 00:21:13.06 so when the autoinducers aren't there, these sensors, LuxN and LuxQ, are kinases. 00:21:18.28 They auto-phosphorylate and the phosphate gets transferred 00:21:22.03 through this circuit to a protein called LuxU and then finally to LuxO. 00:21:26.16 And LuxO's job is to turn off luciferase. 00:21:30.10 And luciferase, which are the genes and the enzymes that make light, 00:21:33.29 is down here. It's called LuxCDABE. 00:21:36.29 So there are five enzymes, C, D, A, B, and E, that are involved in making light. 00:21:41.07 So at low cell density, the system shuts light off. 00:21:44.26 But then at high cell density, so when these autoinducers accumulate, 00:21:48.27 and they get bound by their sensors, it flips a switch. 00:21:51.29 And the sensors change from being kinases to being phosphatases. 00:21:56.15 So phosphate flows backwards through the system 00:21:59.09 and that dephosphorylates LuxO and releases it to allow luciferase to be expressed. 00:22:06.08 And so the bottom line is that at low cell density light is turned off 00:22:10.06 and at high cell density light is turned on. 00:22:13.00 And that matter and the way the bacteria detect those two situations 00:22:16.14 is by whether or not these autoinducer molecules have been built up. 00:22:20.12 And then I want you to also notice that there's another protein 00:22:23.16 which I refer to, named LuxP, that's involved in autoinducer-2 detection. 00:22:28.17 So I just want to remind you that LuxP is the primary binding protein for autoinducer-2 00:22:34.10 and it interacts with LuxQ to send the signal. 00:22:37.12 And LuxP looks like a family of proteins that are typical in bacterial periplasms 00:22:43.07 that all look like sugar-binding proteins. 00:22:46.08 This protein looks like the ribose binding protein of E. coli. 00:22:49.25 And we're going to come back to that in a few minutes. 00:22:51.26 OK, so, by this complicated system information comes in to turn on or off light. 00:22:57.24 And, so, we were curious at this point why Vibrio harveyi would have two autoinducers. 00:23:03.13 So I spent the beginning of my talk telling you about Vibrio fischeri 00:23:06.20 that has this one autoinducer and uses this LuxIR system. 00:23:10.00 What's the point of having two autoinducers feeding into the system? 00:23:14.15 And of course we didn't know the answer to that. 00:23:16.12 But, what we thought was the only way the bacterium can get more out of this kind of circuitry 00:23:21.05 is if those autoinducers encode different pieces of information. 00:23:26.00 Right, if they encode the same kind of information, then two isn't better than one. 00:23:30.21 And, so, we wanted to explore that idea. 00:23:32.26 And, so, what you know already is that these autoinducers are on the outside of cells. 00:23:37.28 And, so, what one can do is just grow up cells, spin the bacteria out of the culture, 00:23:42.28 collect the cell free culture fluids and test them for autoinducers. 00:23:47.00 And so what we did is we just got every bacterium we could get our hands on. 00:23:50.26 We grew cultures of them up, spun them out, and we collected the supernatants. 00:23:54.20 And then we put them onto Vibrio harveyi strains 00:23:57.25 that could only turn on light in response to autoinducer-1 00:24:00.22 or could only turn on light in response to autoinducer-2. 00:24:04.10 So these were engineered strains that could respond to one or the other autoinducer. 00:24:08.19 And what we found in that experiment is that there was never another bacterium 00:24:13.14 that made an activity that turned on light through this first system. 00:24:17.15 But nearly every bacterium that we tested made an activity 00:24:22.14 that turned on light through the second system. 00:24:24.29 And, so, our interpretation of that result is that 00:24:27.13 these two autoinducers are two different languages. 00:24:30.16 Autoinducer-1 is the species specific language for Vibrio harveyi. 00:24:35.00 You'll recall that that's a homoserine lactone. 00:24:37.00 Only Vibrio harveyi makes that. 00:24:39.10 This second autoinducer we could show was broadly made in the bacterial kingdom. 00:24:44.16 We could find hundreds of species that made this activity. 00:24:47.20 And, so, what we thought then is that maybe this is the language of inter-species communication. 00:24:53.02 This is the bacterial trade language. 00:24:55.14 Because if you think about how bacteria really live in the wild, 00:24:59.02 they live in complicated mixtures with hundreds or thousands of other species of bacteria. 00:25:04.02 And if we end up being right in the quorum sensing field that this is about counting, 00:25:08.25 it's not good enough to only be able to count your own siblings. 00:25:12.24 There has to be a mechanism for taking into account other species of bacteria in the environment. 00:25:18.10 And that's what I'm going to try to convince you that autoinducer-2 is about. 00:25:22.03 So to get proof of that, what we did was made mutants that couldn't produce autoinducer-2 00:25:28.02 in several different species of bacteria, 00:25:30.09 including Vibrio harveyi, E. coli, Salmonella, lots of different bacteria. 00:25:34.14 And then we cloned the gene that was involved. 00:25:36.27 And what we found was that indeed, it was the same gene in every case. 00:25:41.16 And so we named that gene luxS. 00:25:45.04 And so what you can do now...you probably know that all these bacterial genomes are sequenced 00:25:50.08 so you can take your gene and plug it into these public databases 00:25:52.27 and then find out who has that gene. 00:25:55.23 And what we found is that, sure enough, at last count there were about 00:26:00.11 500 bacterial genomes that are sequenced 00:26:02.19 and more than half of those genomes contain a highly conserved luxS enzyme. 00:26:07.24 And so what I did was to simply put a smattering 00:26:10.28 of a few of the bacteria that are in the genome database 00:26:13.25 that have this luxS gene. And so what's important about this list 00:26:17.20 is that it is a who's who of clinical pathogens. 00:26:20.24 And so you remember that I told you with autoinducer-1 signaling 00:26:24.09 many bacteria use quorum sensing to turn on virulence. 00:26:28.27 And so what we wondered was that if autoinducer-2 and luxS end up being this generic bacterial language 00:26:35.08 and bacteria are using this to control virulence, one can start to think of making 00:26:40.21 anti-quorum sensing strategies that could be used in a broad range of different bacteria. 00:26:46.02 And so the field now is trying to do that in an effort to make new kinds of antibiotics 00:26:51.27 that no longer kill bacteria but just make them so that they can't count one another 00:26:56.25 and can't turn on virulence. And so what's interesting in that realm is that all of these pathogens 00:27:01.11 and if you look at that list its many, many more pathogens, 00:27:04.09 have luxS. They all make autoinducer-2; we showed that. 00:27:08.00 And what's amazing about the list is that this list 00:27:10.20 is both gram-negative and gram-positive bacteria. 00:27:13.26 So unlike in the acyl homoserine lactone and the peptide quorum sensing systems, 00:27:18.07 we think that autoinducer-2 is a much more ancient language 00:27:21.23 that arose before the split 00:27:23.19 between gram-negatives and gram-positives. 00:27:25.27 So now we knew that all these bacteria made autoinducer-2, 00:27:29.19 they all had this highly conserved gene and enzyme LuxS. 00:27:33.08 And so the questions for us at this point was: What does luxS do in producing autoinducer-2? 00:27:39.29 What is the autoinducer-2 molecule? 00:27:41.12 And then, what do all these other bacteria do in response to autoinducer-2? 00:27:46.07 Obviously they don't turn on bioluminescence. These aren't bioluminescent bacteria. 00:27:50.00 And, so we wondered, do they use this molecule as information? 00:27:53.18 And so that's what I'm going to finish this part of the talk by telling you is first: 00:27:57.22 what is autoinducer-2 and what is luxS's job? 00:28:00.19 And then what these other bacteria are doing with this new molecule? 00:28:04.17 OK, so, the first thing: What's the autoinducer and what's LuxS? 00:28:08.14 So, remember, I've already told you that the bacteria give you this gift. 00:28:11.13 They put these molecules on the outside and so lots of people, you know, 00:28:15.01 we included, have been purifying autoinducers from cell free culture supernatants of bacteria. 00:28:20.07 And so we had been trying to do that with autoinducer-2, sort of doing traditional biochemistry 00:28:25.08 and we could never purify this molecule. 00:28:27.12 And we didn't understand at the time why we couldn't get it 00:28:31.10 but the point was, we couldn't purify it. 00:28:33.05 And so we thought, "OK, we can't use traditional biochemistry to get it 00:28:36.26 but can we use what we've learned from these genomes to just figure out, 00:28:40.15 to infer what luxS does just by looking at where it's placed in these genomes?" 00:28:45.12 So probably you know that bacteria put genes that work in pathways 00:28:50.05 nearby one another, usually in operons. 00:28:52.10 And so what we did was to just look over these genomes and ask: 00:28:56.01 "Could we find any pattern to where luxS was in these different genomes?" 00:29:00.21 And in a couple of cases, indeed, we could. 00:29:03.03 And so I've just put on this next slide the case that we've found for Borrelia burgdorferi, 00:29:03.17 which is the bacterium that causes Lyme disease. 00:29:09.20 What we noticed was that LuxS was in a three gene operon in the Borrelia genome 00:29:15.25 and in a few other genomes. 00:29:17.15 And the operons were always set up like this. There were three genes. 00:29:20.24 One was called pfs, the second one was called metK and then luxS was right next door. 00:29:26.16 And so we didn't know what luxS does, but in fact, pfs and metK have known functions. 00:29:33.02 And they work together in a biochemical pathway. 00:29:35.28 And so we wondered, "Couldn't luxS work in that pathway with pfs and metK?" 00:29:40.29 And so what has been known about pfs and metK for almost 50 years now 00:29:45.19 is the following: those two enzymes work in S-adenosyl methionine utilization. 00:29:51.21 So maybe you know that S-adenosyl methionine or SAM is a very important substrate in cells. 00:29:57.23 It's involved in methylating different substrates. 00:30:00.04 So SAM is involved in putting methyl groups on DNA and RNA and proteins. 00:30:04.25 So you can't make any of those things unless you have SAM. 00:30:07.17 So it's a really important, ancient molecule. 00:30:10.06 And so the way that that works is that methionine is converted into S-adenosyl methionine 00:30:16.06 by that first enzyme we noticed which is called metK. 00:30:19.10 And then S-adenosyl methionine or SAM does its job. 00:30:23.08 It puts a methyl group on all these different substrates. 00:30:25.19 And so what the cell gets from that is the thing it wants, 00:30:28.18 like DNA or RNA, but it gets, unfortunately, a bi-product which is called S-adenosyl homocysteine. 00:30:35.27 And this is incredibly toxic because it feed back inhibits all of these methyltransferases 00:30:41.10 that are putting the methyl groups on the different substrates. 00:30:44.17 So what happens is that every time SAM methylates something a molecule of SAH is produced 00:30:50.06 and the cells have to get rid of that because of its toxicity. 00:30:53.25 So the way that they decrease toxicity is with this second enzyme I told you about, pfs. 00:30:59.24 So pfs is a nucleosidinase that takes adenine off of SAH and makes S-ribosyl homocysteine. 00:31:07.15 And so that relieves toxicity. 00:31:09.25 And it's been known since the early 60's that S-ribosyl homocysteine gets converted by 00:31:15.14 some unknown enzyme into homocysteine 00:31:18.02 and this funny molecule, 4,5-dihydroxy-2,3-pentanedione. 00:31:22.01 that nobody knows what it does. 00:31:23.15 And so what we wondered when we saw that metK and pfs were involved in this pathway 00:31:28.12 and there was a step that didn't have an enzyme associated with it 00:31:31.27 we wondered, "Well, couldn't that be luxS? 00:31:35.01 That what luxS's job is to do is to carry out this last step in this pathway." 00:31:40.10 And so to prove that was the case, what we did is we cloned and purified the luxS protein. 00:31:45.26 We put that protein in a test tube with the molecule S-ribosyl homocysteine, 00:31:50.27 let it react and then we filtered out the products. 00:31:53.16 And what we got from that was homocysteine and autoinducer-2. 00:31:58.07 And so indeed this is the pathway for making autoinducer-2. 00:32:02.03 And this final product that nobody had ever known what it was, is in fact, autoinducer-2. 00:32:07.16 And so this has always been considered a salvage pathway in bacteria. 00:32:12.16 They have to detoxify to get rid of this SAH 00:32:15.22 and doing it this way they get two useful things: they get adenine at the first step 00:32:20.11 and they get homocysteine at the second step. 00:32:22.23 But we think the real reason this pathway has evolved is because what they're trying to make 00:32:27.09 is this funny molecule which I'm trying to tell you 00:32:29.15 is a molecule involved in interspecies communication. 00:32:32.20 OK. So now I'm going to show you the biochemistry that luxS does. 00:32:37.21 So this is the reaction. So this is the molecule S-ribosyl homocysteine. 00:32:42.06 And the enzyme luxS's job is to just cut that in half 00:32:45.24 into homocysteine, which gets recycled, and then this molecule that I told you about 00:32:50.16 which is called 4,5-dihydroxy-2,3-pentanedione. 00:32:54.09 So we knew we could do this experiment and we knew we had autoinducer-2 activity in it. 00:33:01.00 So the guess was that this is autoinducer-2. 00:33:03.26 And so what we did to prove that was we carried out big reactions. 00:33:08.01 Right? Then we filtered out the protein 00:33:09.25 and then we took what was on the bottom of those reaction mixtures to the chemistry department 00:33:14.20 and did mass spec and NMR and techniques like that to try to find this molecule. 00:33:19.11 And what was interesting during this experiment is that we could find lots of homocysteine 00:33:24.12 in these reactions but we could never find this molecule. 00:33:27.21 So even though we knew the biochemistry, 00:33:29.23 we had one enzyme and one substrate in a test tube, 00:33:32.18 we couldn't find the product. 00:33:34.01 And the reason is because this molecule, this pentanedione, is incredibly reactive. 00:33:39.15 What happens is that as soon as it gets made, it starts cyclizing 00:33:43.09 into family of molecules that are in a fast equilibrium, all of which are derived from this precursor. 00:33:49.22 So you get many molecules and they're all sort of the same thing 00:33:52.17 because these rings are just opening and closing. 00:33:54.24 And so what we thought then is that one or some of those molecules 00:33:58.26 must have the autoinducer-2 activity, 00:34:01.15 but we still didn't know the structure. 00:34:04.02 And so actually, I have to say that we were a little discouraged by that because 00:34:07.05 here, we could do the reaction in one step, but we still didn't know what this elusive molecule's structure was. 00:34:12.13 And so we knew we couldn't get it from this because these molecules are opening and closing 00:34:16.25 really fast and they're all turning into one another. 00:34:19.10 And so the question was: how could we figure out which was the molecule that carries the activity? 00:34:24.09 And so what we decided to do is to just go back to what we knew about how 00:34:29.03 autoinducer-2 activity was detected. And you'll remember that I showed you 00:34:33.05 this cascade for how the information from these autoinducers comes into the cells. 00:34:37.25 And you might recall that I pointed out that there were two proteins 00:34:40.25 involved in autoinducer-2 detection, LuxP and LuxQ. 00:34:45.15 And what I slipped by you earlier in my seminar 00:34:48.07 is that this protein, LuxP, is a periplasmic protein that looks like the ribose binding protein. 00:34:54.06 And what I just showed you on this slide, 00:34:57.05 is that LuxS actually takes the ribose moiety of S-ribosyl homocysteine 00:35:04.13 and opens it up. But when this molecule closes back into a ring 00:35:08.09 it looks a little bit like ribose because it was made from ribose. 00:35:11.28 And so that started to make sense to us, that there should be 00:35:15.05 a protein that looks like it can bind a ribose-like molecule involved in autoinducer-2 detection. 00:35:20.20 So what we decided to do then was to take this protein, and purify it. 00:35:25.28 And get it to reach into this in vitro mixture that has all of these molecules 00:35:29.29 that are made from the pentanedione. We could get it to reach in there 00:35:34.19 and pull out the rearranged moiety that had the autoinducer-2 activity. 00:35:39.04 So sort of like an affinity purification. 00:35:41.20 Of all this mix of molecules that were rearranging in solution, just let LuxP 00:35:46.16 reach in there and grab the one that had...that is autoinducer-2. 00:35:51.06 And so we did that experiment in collaboration with a colleague 00:35:54.26 at Princeton University; a professor names Fred Houston, who's a crystallographer. 00:35:59.10 And so what we did was to crystallize LuxP 00:36:01.21 in the presence of autoinducer-2 and solve the structure. 00:36:04.26 And in fact that turned out to be pretty easy to do 00:36:07.16 because these periplasmic binding proteins are very well studied 00:36:13.05 and they all look sort of the same. So this is our crystal structure of LuxP. 00:36:17.25 And all of these proteins, including LuxP, look sort of like clam shells. 00:36:22.03 They lock down on the middle to a sugar that's at the interface between the two domains. 00:36:28.05 And our protein was no different. It looked like this clam shell 00:36:31.09 and there was a big space in the center where a molecule could bind. 00:36:35.11 And when we did a little closer up version of this, 00:36:38.11 where we just get rid of all the electron density from the amino acids, 00:36:42.28 what you're left with is more or less a skeleton of the crystal structure 00:36:47.21 and right at the ligand binding domain, we could see a lot of electron density 00:36:52.13 indicating that the autoinducer-2 molecule was there. 00:36:55.13 And so to prove that that was correct what we did was we just heated the protein up to 00:36:59.19 about 50 degrees and let this stuff fall out 00:37:02.25 and if you put it in a tube with Vibrio harveyi, they turn on light. 00:37:06.08 So indeed, this was the autoinducer. 00:37:09.05 So we could zoom in a little bit more to try to figure out 00:37:11.29 what was the molecule that was sitting there in the active site. 00:37:15.10 And so in this slide what you're looking at is the amino acids from LuxP 00:37:21.00 that coordinate the ligand. And I hope what you can see are two rings. 00:37:25.25 And this was a surprise to us because from the biochemistry 00:37:28.21 I showed you a couple of slides ago, when that pentanedione molecule folds up 00:37:33.18 there's only five carbons in that. 00:37:35.24 And we could account for those five carbons from this bottom ring. 00:37:39.15 So this was the folded up pentanedione that we knew about. 00:37:42.19 The problem was there were all these other atoms on the top. 00:37:45.29 And we were confused by that because we didn't know where they came from. 00:37:50.08 So to prove in fact that this was the correct molecule what we did was a mass spec of the protein 00:37:56.08 bound to the ligand and then we did this gentle heat denaturation so that the ligand falls out 00:38:01.12 and we took the mass of the protein again. 00:38:03.29 And the difference in mass is 194 which is exactly what this molecule weighs. 00:38:09.09 And so what we thought then is that LuxS, and we know this now, 00:38:13.06 every LuxS makes that linear pentanedione and then it folds up into this ring structure 00:38:19.12 but they're still very reactive because these rings have a lot of oxygens on them 00:38:24.00 which are highly reactive. So we thought, "OK, something must add to the top across those oxygens 00:38:30.08 to make this double ringed molecule that has a molecular weight of 194. 00:38:35.20 And the surprise was this atom right here. 00:38:39.13 We thought that should be carbon because that would connect the ring 00:38:42.15 and that would make a molecule of 194. 00:38:44.21 The problem was that chemists didn't like that idea because it's very well known 00:38:48.21 that carbons are very unstable if they have four oxygens on them. 00:38:53.10 And so what we found out later was, in fact, that's not a carbon atom. 00:38:57.01 What it is is a boron atom. 00:38:59.10 So it turns out that boron is very abundant in the ocean. 00:39:04.25 Right? And remember, this is a marine bacterium. 00:39:07.20 And boron also loves to add across cis-hydroxyl groups that are on rings. 00:39:13.12 And so what we found out, in fact, was that the real signaling molecule 00:39:17.05 is this one that I'm showing you here, 00:39:19.09 that has a boron on the top of that ring. 00:39:22.00 And to prove that that was the case we did boron NMR. You can look for boron 11. 00:39:27.05 And showed that, sure enough, this is the Vibrio harveyi molecule and it has boron in it. 00:39:32.12 And so that was actually a surprise because boron, 00:39:34.23 even though it has a very well studied role in chemistry, 00:39:37.13 it doesn't have a very well defined role in biology. 00:39:40.17 But in fact there's lots of it the ocean 00:39:43.01 and so this molecule could be made spontaneously 00:39:46.00 when that pentanedione gets released from the bacteria. 00:39:48.25 And so now, in fact, even though this molecule doesn't look like the biochemistry 00:39:52.21 that I showed you from the LuxS reaction, in fact, we can account for everything that's happening. 00:39:57.14 What we know now is that every LuxS protein makes this molecule, which we call for short DPD, 00:40:04.28 for dihydroxy pentanedione. Every LuxS makes that linear pentanedione. 00:40:10.22 But that molecule, as I told you is very reactive, and so it starts cyclizing. 00:40:14.19 And in the case of Vibrio harveyi, this important ring is formed, 00:40:18.13 and you have to notice that this is a chiral carbon with the oxygen up and the methyl down. 00:40:23.01 So this ring gets formed. That gets hydrated spontaneously. There's a lot of borate in the ocean 00:40:28.19 which adds across these two oxygens to make the final signaling molecule 00:40:32.28 that we found in the crystal structure. 00:40:35.03 And so what we know is that all of these reactions happen spontaneously 00:40:39.04 based on the precursor molecule that the bacteria make. 00:40:42.05 And, in fact, we can now make this molecule DPD. 00:40:45.15 We can put into a test tube and we can watch all of this happen. 00:40:49.01 And so we're very confident now about how 00:40:51.13 the Vibrio harveyi...what molecule Vibrio harveyi 00:40:54.12 uses for quorum sensing, autoinducer-2 quorum sensing. 00:40:58.24 But once we figured this out it actually gave us another puzzle. 00:41:03.01 Which is, as I've told you, there's a lot of boron in the ocean, 00:41:05.28 so maybe this kind of a molecule makes sense for Vibrio harveyi. 00:41:09.04 But I've also told you that hundreds of species of bacteria make and use autoinducer-2 00:41:14.00 and there's very little boron in terrestrial environments. 00:41:16.23 And so what we wondered, "Is autoinducer-2 one word, this one, 00:41:22.00 or is it a family of words that are all made from this precursor?" 00:41:25.11 So for example, this could be someone's autoinducer, 00:41:28.01 or this could be another bacteria's autoinducer. 00:41:30.15 And the other thing that I alluded to a minute ago is that there's this chiral carbon. 00:41:34.19 So when this molecule folds up, oxygen up, methyl down, there's another pathway 00:41:39.24 which is the opposite stereochemistry that could be across the bottom of this chart. 00:41:44.16 So there's many molecules that are possibilities. 00:41:46.28 And we wondered, "Are bacteria using all of these other molecules as autoinducers?" 00:41:51.26 And so we wanted to try to do experiments to get at that 00:41:54.22 and of course the one thing that we had learned from this 00:41:57.17 is we couldn't just purify molecules from cell free supernatants 00:42:00.18 because all of these are present. And so what we had to do is a trick similar to one 00:42:05.03 that I've shown you which is to get a sensor from some bacteria, 00:42:09.00 get it to reach in and pick out the molecule it likes. 00:42:11.28 And so, what was lucky was at the time that we were working on the chemistry of autoinducer-2 00:42:16.22 we were also trying to answer another question that I alluded to earlier 00:42:20.16 which is: What do other bacteria use autoinducer-2 to do? 00:42:25.16 What genes do they turn on and off? 00:42:27.07 And so we had started to work on E. coli and Salmonella, enteric bacteria. 00:42:31.27 And simply, we were doing genetic screens to ask, "What are the genes 00:42:35.15 that are controlled by autoinducer-2 in E. coli and Salmonella?" 00:42:39.05 And we found a number of genes that autoinducer-2 regulates 00:42:42.15 but what's important for today's talk is that we found this operon 00:42:46.07 that we named Lsr for LuxS Regulated operon. 00:42:50.07 And this operon is in the E. coli and the Salmonella genome 00:42:54.17 and it's annotated as the ribose-like transporter operon. 00:42:58.06 So what the Lsr operon encodes is what's called an ABC transporter. 00:43:03.07 And ABC transporters are widespread in different organisms 00:43:07.03 and their job are to bring molecules from the outside in. 00:43:10.18 And so they all sort of look like this one. 00:43:13.07 What they have is a B component which is a binding protein 00:43:16.26 that binds some ligand and takes it to the channel. Right? 00:43:20.07 And delivers it and it comes across into the cell and the energy is supplied by the A component 00:43:26.01 which is an ATPase. And so we found that autoinducer-2 induces the genes 00:43:32.05 that make this transporter operon and this protein LsrB 00:43:36.16 which is a binding protein, looks just like LuxP from Vibrio harveyi. 00:43:40.23 It looks like a ribose-like binding protein. 00:43:43.17 And so, of course, what molecule do we know that looks like ribose? 00:43:46.13 Well, it's autoinducer-2. 00:43:48.07 And we had already shown that the way E. coli and Salmonella respond 00:43:51.10 to autoinducer-2 is that they put it out. Then they bring it in with this transporter 00:43:56.23 and then they respond to it on the inside. 00:43:59.01 And so, of course then the guess was that the molecule that LsrB 00:44:02.28 would be binding in E. coli and Salmonella was their autoinducer-2. 00:44:07.07 And so, exactly analogous to what I told you about a few slides ago, 00:44:10.21 We crystallized this protein, the Salmonella LsrB protein, with the ligand 00:44:15.07 and solved the structure. And what we found was that 00:44:19.03 as expected, the protein looked very similar to LuxP and again, 00:44:23.09 the ligand was in the ligand binding site. 00:44:26.03 And so we could just solve the structure and what we found was that, indeed, 00:44:29.15 the Salmonella molecule and the Vibrio harveyi molecule are different molecules. 00:44:36.07 So now what I have done is to put the two molecules side by side. 00:44:41.00 And so you'll remember that the Vibrio harveyi molecule is this double ringed molecule 00:44:45.15 that has this boron moiety at the top of the ring. 00:44:48.18 The Salmonella molecule, what you can see is that it doesn't have that boron ring. 00:44:53.16 And that makes sense to us because there is no boron in your gut. 00:44:57.03 There's hardly any boron there. Salmonella would never find the molecule that looked like that. 00:45:01.04 So the first thing that's different is that Salmonella's autoinducer-2 doesn't have boron, 00:45:06.09 which makes sense given it's a terrestrial environment 00:45:08.12 and Vibrio harveyi is a marine environment. 00:45:10.20 And the second thing, which is a little more subtle, that you have to notice 00:45:13.20 is that this is that chiral carbon; oxygen up, methyl down in the case of Vibrio harveyi. 00:45:18.26 In the case of Salmonella, the stereochemistry is the opposite; methyl up, oxygen down. 00:45:24.10 But in fact, even though these molecules look different, we can connect them really easily. 00:45:29.01 And so, you'll remember the chemistry that I showed you about Vibrio harveyi 00:45:33.28 is that all the LuxS make this pentanedione and then by these reactions I showed you 00:45:38.19 and borate addition, you get the Vibrio harveyi signal. 00:45:41.23 In the case of Salmonella, remember the other 50% of the time 00:45:45.24 that the DPD molecules cyclize, 00:45:52.22 remember 50% of the time they're going to cyclize with the methyl up and the oxygen down. 00:45:57.07 That's important for Salmonella. That molecule gets hydrated 00:46:01.10 and this is the molecule that we find in the Salmonella ligand binding pocket. 00:46:05.15 And so now we know only two autoinducers that are made 00:46:09.16 from this precursor molecule, autoinducer-2. 00:46:12.07 And so of course, we're still looking to find if 00:46:14.04 this one's an autoinducer or this one or this one. 00:46:16.11 Right? But that we haven't done yet. But even though we don't know 00:46:19.13 the complete lexicon of how these bacteria use the information in that molecule to talk, 00:46:24.02 we do understand the principle for how interspecies communication works. 00:46:28.13 Because all of these molecules are freely rearranging in a very fast equilibrium 00:46:33.27 Salmonella can talk to Vibrio harveyi and likewise Vibrio harveyi can talk to Salmonella 00:46:38.17 because their molecules rearrange in between the sender and the receiver. 00:46:42.24 And so for example, we have done experiments to show if you put a lot of boron 00:46:48.10 in this reaction mix, all of the equilibrium goes to the Vibrio harveyi molecule. 00:46:52.23 Vibrio harveyi's quorum sensing response turns on and Salmonella is inhibited for quorum sensing. 00:46:58.07 Likewise, one can chelate all of the boron out of the medium and then the equilibrium 00:47:03.00 shifts to this way and Salmonella starts chit chatting and Vibrio harveyi can't talk. 00:47:07.26 So even though we don't know all of the molecules involved, 00:47:11.14 we understand that the reason interspecies works through this interconverting molecular system 00:47:16.06 is because this precursor is common for all of these bacteria and so, in fact, 00:47:21.11 I could talk to you and you can talk to me because they depend on these fast equilibrium reactions. 00:47:27.15 And so that's the state of affairs. 00:47:29.13 We're still trying to find more of these autoinducers that different bacteria use. 00:47:33.27 And so now I've told you two of the three things that I said. 00:47:36.23 What LuxS does: LuxS makes this pentanedione. 00:47:40.13 What is autoinducer-2? Well, we've started to understand that for 00:47:43.28 a few of these species and we think that there are more molecules involved. 00:47:47.01 But we understand how they talk between species. 00:47:50.00 And then the last thing that I said I would tell you is: What are other bacteria doing with autoinducer-2? 00:47:55.19 And so this is just a list of some of the bacteria 00:47:58.01 that different people in the field have started to study. 00:48:00.26 And so what you can see, this is just the name of the different species of bacteria 00:48:04.27 and then on this side are the genes that different colleagues of mine 00:48:09.01 have found that are controlled by autoinducer-2. 00:48:11.25 And so if you actually tried to read this list, what you can see 00:48:15.01 is that in most every case that's been studied, autoinducer-2, 00:48:19.16 just like peptides and homoserine lactones, controls biofilm formation and virulence. 00:48:25.09 And so it appears that the quorum sensing really is a big player. 00:48:29.11 Both intra-species and interspecies quorum sensing 00:48:32.15 are big players in controlling virulence and biofilms. 00:48:36.16 And so where the field is moving now is for people to either take their favorite autoinducer-1 00:48:41.29 or generically, to take autoinducer-2 and try to inhibit the synthases or 00:48:46.29 inhibit reception of those molecules to get new kinds of antibiotics. 00:48:51.11 So, when one works on an autoinducer-1, 00:48:53.20 the hope is to make a species specific antibacterial therapy. 00:48:57.27 And of course the hope for autoinducer-2 is that we could get an inhibitor that controls virulence 00:49:04.13 in all kinds of bacteria because you'll remember, again, both gram negative bacteria 00:49:08.07 and gram positive bacteria use autoinducer-2 quorum sensing to communicate. 00:49:13.15 And so that's sort of the practical way the field is going. 00:49:16.17 But I think we've learned a lot about chemical communication and also about 00:49:20.27 how bacteria have developed multicellularity. 00:49:24.05 And so to sum up, I hope what you've learned through this first part of the talk 00:49:27.27 is that bacteria can talk to each other and their languages are chemical. 00:49:32.08 They don't use words and sounds the way we do, they use chemicals. 00:49:36.05 We know that beyond being able to talk to one another, 00:49:39.20 that they can have multiple languages. And so at a minimum, 00:49:46.14 by having an autoinducer-1 and an autoinducer-2, what we now understand is 00:49:51.05 that bacteria can speak both within and between species. 00:49:56.06 So they have intra- and inter-species communication. 00:49:59.16 We also now know that by just having those two molecules, 00:50:04.09 what that means, we would argue, is that bacteria can distinguish self from other 00:50:09.17 by using a private language and a community language. 00:50:12.26 And what we think, then, is, of course, that's what happens in your body. 00:50:15.29 Its not like your kidney cells get all confused with your heart cells every day. 00:50:19.11 And that's because they use different hormones or different chemicals 00:50:22.14 to communicate and let these different organs in your body carry out different functions. 00:50:27.20 Again, bacteria have been on this earth for billions of years. 00:50:30.20 We think that they invented the idea of distinguishing self from other. 00:50:34.24 The other thing that we know is that there's many more molecules to be discovered. 00:50:40.21 This field is only 10 or 15 years old. We're at the very beginning of it. 00:50:44.27 It's a really exciting time. What I hope you've learned from today's talk 00:50:48.10 is that, indeed, bacteria can distinguish self from other, 00:50:51.17 but autoinducer-2 is a generic molecule. 00:50:54.13 So it doesn't say who the other is. And we know when bacteria make 00:50:58.16 complicated, architected communities like in biofilms 00:51:02.00 in fact, these are very ordered structures, you know, with one species next to another 00:51:06.17 next to a third. They're not willy-nilly. 00:51:08.12 And so we believe, the only way bacteria can get that is to have molecules that say 00:51:13.07 who the other guy is. And that's what this field is hunting for now. 00:51:17.05 And I think that's what you'll be hearing about in the next few years. 00:51:20.07 And then what I've argued and I hope that you think is an interesting idea 00:51:25.12 is that quorum sensing let's bacteria be very similar to higher organisms. 00:51:30.21 Bacteria don't act as individuals. Often times they need to act as a community, in synchrony. 00:51:37.14 And by communicating with chemicals, this allows bacteria to carry out 00:51:41.15 traits that are very similar to those carried out by higher organisms 00:51:45.02 because they've established this way to control gene expression instead of over a single cell, 00:51:50.14 over the entire population. And, of course, along with learning about these first things, 00:51:56.02 we now understand that there's a lot of opportunities for novel biotechnological applications. 00:52:01.02 For example, to make, as we've discussed, to make new therapies, new antimicrobial therapies. 00:52:06.17 Also, people would like to put these anti-quorum sensing molecules in plastics. 00:52:11.08 If one knew one was going to go to the hospital and get a catheter 00:52:13.28 that they'd get infections. If you could embed these in paints or plastics or implants 00:52:18.13 or the saran wrap that wraps up your meat at the grocery store it would be really great if 00:52:23.04 we could just use these anti-quorum sensing molecules to fight against pathogenic bacteria. 00:52:28.17 And at the same time, of course, we now know that we are covered with bacteria. 00:52:32.24 Bacteria live in us and on us all the time and they're playing an active role 00:52:36.06 in keeping us healthy. So we'd also like to look for pro-quorum sensing molecules, 00:52:40.27 molecules that make quorum sensing better in beneficial bacteria... 00:52:46.16 that help to fight off invaders. 00:52:48.00 And so we're looking for both agonists and antagonists of quorum sensing molecules 00:52:53.04 for novel biotechnological purposes. 00:52:55.23 And the final thing to do is to make a confession 00:52:58.14 that I'm not so smart. These bacteria have already figured this out. 00:53:01.15 They have a billion year head start on us. 00:53:03.02 What the field has been learning which is really exciting in the last couple of years 00:53:06.28 is that there's all kinds of natural anti-quorum sensing strategies 00:53:11.00 happening within the bacterial populations themselves. 00:53:14.00 So there's bacteria that eat each other's auotinducers. 00:53:16.07 There's bacteria that cut each other's autoinducers in half. 00:53:18.26 They eavesdrop. They cheat. They free-ride. They're doing all the kind of things that 00:53:22.26 we'd like to do and so we think that we can get a lot of the hints for how 00:53:26.05 to make these strategies just from the bacteria themselves. 00:53:29.12 So, thank you for listening to this talk. 00:53:31.25 The second half will be a much more practical talk but that's the beginning 00:53:35.28 and I hope you think, by now, that bacteria can talk to each other. 00:53:38.27 And, again, I'm Bonnie Bassler from Princeton University.