 All right, we're going to get started. It's my great pleasure to introduce Professor Bassler for the Steambach Lectures of Biochemistry. To honor Professor Steambach, there is a two-part lecture series that takes place every year. And he had numerous contributions to the field of biochemistry and also nutrition. Dr. Bassler received her bachelor's in biochemistry from UC Davis and her PhD in biochemistry from Johns Hopkins. She is the Squibb Professor of Molecular Biology at Princeton University and a Howard Hughes Medical Institute investigator. Her research focuses on understanding the molecular mechanisms of bacterial communication and interspecies communication. So quorum sensing allows cells, single cells, to synchronize their behavior on a population-wide scale. And it controls virulence, biofilm formation, antibiotic production, and may also constitute a novel mechanism for antibiotics or antimicrobial therapy. Among her many important contributions, she actually discovered the structure of auto-inducer 2, which is a universal interspecies quorum sensing molecule. Dr. Bassler has won numerous awards and recognitions, and I won't be able to list all of them here. Otherwise, I'll be here for a very long time. So some highlights include she is a member of the National Academy of Sciences and the American Academy of Arts and Sciences. She was a MacArthur Fellow in 2002. She won the Wiley Prize in Biomedical Science in 2009. The L'Oreal UNESCO for Women in Science Awards in 2012, and also the Shaw Prize in 2015. So as a reminder, there will be a reception in the Atrium of Biochemical Sciences building across the street, which I hope to see everyone there. And Dr. Bassler will have another seminar at this time in Ebling tomorrow, and her title is How Bacteria Distinguished Self from Other. And I hope to see everyone there as well. So please welcome Dr. Bassler. Well, thanks, Julia, for that lovely inflated introduction. Can you guys hear? And then also, I just want to thank everybody. I've had just a magical day, and I'm looking forward to more magic tomorrow. And it's such a privilege to get to give these lectures in the spirit that this seminar series was begun, and the lineup of people who've given this lectureship is kind of intimidating. But I'm glad to be one of them. And so anyway, I just thank you for spending your time with me. I thank you for telling me your science stories, and I hope to return that favor twice to you by telling you about two different projects, one today and then a different one tomorrow. Let's see. So then I will get going. One more time, heartfelt thanks. I know it takes a ton of work to put these things together. But I've enjoyed it so far. OK, so let's see. So let me get going. You've already gotten a hint. I want to first just give a little background and try to convince you the bacteria can talk to each other with a chemical language, and that that allows them to do tricks that they couldn't do as individual cells. And then I want to try to move on during my talk to try to show you that by learning about these languages, the bacteria used to communicate and to coordinate group behavior, that maybe if you learn enough about it, you can do something practical, which is to try to think about how to manipulate these conversations and get the bacteria to do things on demand. And so that's sort of where we're going in today's talk. OK, so to give some context, for 150 years, bacteria had been known to cause disease. And so on the top of this slide are just some pictures of some notorious pathogens, just pictures of bacteria taken under a microscope that caused these debilitating or deathly diseases. So that's been known for 150 years that there's these bacteria that shouldn't be in us or on us. And when they do get in or on us and get a toehold, they can bring us onto our knees or they can kill us. And that's true for animals as well as plants. But then increasingly, what we've been learning in the past just few decades is that actually bacteria perform all these miracles for us as well, which what we now know is there's this magical consortium of bacteria that we call the microbiome that lives in and on all higher organisms. And it gives those organisms genes that their own genomes don't have. And so we're only now learning about all of the benefits of the microbiome, but in the case of humans. In some cases, some things that we already do know about is so for example, we don't have any of the enzymes that allow us to access nutrients from plant food. So we need these bacteria in our guts to give us those calories. They educate our immune system in our early life to keep bad microbes out and allow good microbes in. They make many of our micronutrients. This list is going on and on and on. But what is really clear is that there would be no life on Earth if it weren't for these bacteria. So they are, on the one hand, can take our lives away. And on the other hand, they give us our lives. So those are just facts at this point. And so the question in my gang's part of this business of these amazing things that bacteria can do is not the fact that they can do it. But what we always try to think about is how do they do it? These bacteria are so tiny. How do they get the power either to kill us or to give us our lives? So what we believe is that much of that power comes from this process of communicating and coordinating behavior so that the bacteria act in groups to be able to carry out tasks that they could never accomplish as individuals because they're too small. And so that process, we now have a name for, and we call it quorum sensing. And so what my field now thinks is that bacteria live in two different modes. They're either alone and acting, going solo, or they're in groups, and they're acting as part of communities. And so they live in those two kinds of different ways, but they have to know which of those situations they find themselves in, and then they have to act appropriately based on whether they're alone or whether they're in a group. And so the way that they do it is with a chemical communication process. And so in this sort of simple cartoon, this is supposed to be my cartoon of a bacterium. And so when bacteria are at low cell density, so when they're alone, they want to have the program of gene expression going that's good for acting as an individual. So they're carrying out some subset of the task that they're capable of doing. And among the things they do is that they make and release small molecules that we call auto-inducers, and I have depicted by these red triangles in this cartoon. And so since the world is big and bacteria are small, at low cell density, the auto-inducers diffuse away. The bacteria can't detect them, and that says behave as an individual. But then as the bacteria grow and divide, since all of the cells produce a share of the auto-inducer molecule, the concentration of the auto-inducer increases in proportion to cell number. And so what happens is when the auto-inducer hits a particular threshold amount, the bacteria detect it, and they infer from that detection event that they must have neighbors around. So in unison, all the bacteria change their gene expression, and they begin to carry out group or collective behaviors. And so in fact, the bacteria don't have any idea of whether there's cells next to them. They're using this chemical as a proxy for cell number. And we know we're right about that, because we can spin the cells out of solution, and if we just add the molecule to the dilute cells, they'll do all the group behaviors. So they're just measuring the chemical to infer the number of bacteria around. And so quorum sensing is widespread in the bacterial world. There are thousands of examples of it by now, but actually the reason that I drew these bacteria in blue is because it was originally discovered by Woody Hastings in a bioluminescent marine bacteria that just turned on light in response to quorum sensing. So it turned on this beautiful blue bioluminescence, this marine bacteria. And so now what we know is that in every bacteria that's been studied, hundreds of genes, up to 600 genes, so a quarter of the bacterial genome, can be under these quorum sensing circuits. But most of those behaviors are invisible. And so why I think this got missed for so long is because whether bacteria are acting alone or acting as part of a group, how would you know? You can't see them. You can't see their behaviors. But what was so amazing about bioluminescence and the way in for Woody was that these bacteria made perceivable light. And so you could see that these bacteria did this together. And so that, for the first time for the scientists, made the invisible world of the bacteria visible to us, and then we could follow it and monitor it to figure out how this process occurs. But now what we get, fast forward 25 years or so, and now what we get is that this is broadly in the bacterial world, and as you already heard, it controls all kinds of group behaviors like biofilms, virulence, factor production, antibiotic production, mating, all kinds of things where you need a lot of bacteria working together for the task to become successful. But I drew them in blue because this is a special seminar series, so I wanna go back to my roots and I'm gonna tell you about Vibrios today. So everything I'll tell you about comes from these first systems that where quorum sensing was discovered. And so I'm gonna tell you the very particular components of the Vibrios circuit, and so they will be particular, but what I wanna try to tell you is that the principles that I tell you, even if the bacteria tinker with the particular components, the principles that I'll tell you about transcend all quorum sensing systems. So the rules that I'll give you are the same even if the components change. Okay, so what about this language? So what we now understand is that there have to be at least three quorum sensing words, molecules, to have an appropriate conversation. So what we know is that each, what we think is that each species of bacteria makes some molecule. In the case of this Vibrio, this green, homocerylactyl molecule, some molecule that it and only it makes. And so this molecule is for intrus species communication. As it builds up, it allows the bacteria to say, this is how many of my clones are around. And then there's a molecule we discovered, this purple molecule, this fatty acid that all Vibrios make, but nobody but Vibrio makes based on genome sequencing and sampling experiments. So we think this is the language of the genus, the Vibrio language. So this, the green molecule says you're my twin, and the purple molecule says you're my cousin. And then there's a molecule that we discovered that is made by half of all bacteria that have ever been tested, gram negatives and gram positives, this orange molecule, and they all make the identical molecule. So we think there's no species information in this orange molecule. It's for inter-species, across-species communication. So those are the examples of the three molecules. And what we think the bacteria do, I think that's what they're trying to do, the computation is as these molecules build up, the first thing the bacteria ask, they say, am I alone or am I in a group? So they measure the amounts of these molecules and they start turning on and off genes. But then the more sophisticated computation that we've recently shown they do is that they actually measure the ratio of these molecules. And then what I think they're asking is not just how many bacteria are around me, they're asking who is around me. And so there's species information encoded in these molecules, so the bacteria can both ask how many and who is my neighbor? And then they change what genes they turn on and off based on whether they and their kin are in the majority or, for example, if it's the enemy. So I think that's what they're trying to do with these molecules. So that's about the molecules. And now how does the information go from the outside in? So this is supposed to be the cell membrane and these molecules each have a dedicated receptor that sits in the membrane. And so at low cell density, when there's no auto-inducers there, these receptors are kinases. And they autophosphorylate and they send phosphate through this integrator protein to this called Lux-U, to a protein called Lux-O. So at low cell density, Lux-O is phosphorylated and that activates it. And what Lux-O is is a transcription factor. And so the phospholuxo turns on the expression of a set of small RNAs. And so for those of you who work on eukaryotes, these are like the microRNAs of eukaryotes. So at low cell density, these microRNAs get made and then what they do is they sit on the mRNA encoding a protein called AfA. They reveal the ribosome binding site and so this AfA protein gets made and that's good because this is the master transcription factor that runs all of the individual behaviors. The small RNAs also act negatively and so they sit on the mRNA of a protein called HAPR and they repress it, that message gets destroyed and so no HAPR gets made and that's good because it runs the group behavior program. So at low cell density, AfA gets made and the bacteria act as individuals. Then as the bacteria grow and divide, the auto-inducers are accumulating and when they reach their critical amount, they interact directly with their receptors. And so when the receptors bind the auto-inducers, it flips a switch and the receptors change from being kinases to being phosphatases. So now phosphate flows backwards through the circuit. Lux-O is dephosphorylated, it's completely inactive under that condition so these smaller RNAs aren't made. So now AfA isn't activated but HAPR isn't repressed. So at high cell density now HAPR is made and it governs the group behavior program. So it's actually a really simple circuit. One of these two master transcription factors is made at low cell density and high cell density and that is dictated by whether or not those auto-inducers are there and that's by whether or not the bacteria are in a group or they're alone. So that's how the circuit works. And so we discovered everything that you see on this slide and we like to study all of the levels that you see there. We like to understand what are the molecules? How do the bacteria detect them? How do they not get confused by these different blends of molecules? We try to understand how does that information get integrated into the circuit? And then we wanna understand what does it mean for a bacterium, what are the behaviors? What does it mean for a bacterium to act as an individual versus to act as part of a member of a community? And so we try to understand all of those features. And so what I thought I would do today is to just tell you two stories that sort of give you a flavor of those kinds of investigations that we undertake. So the first story is gonna be about molecules and the circuit and then the second story will be about quorum-sensing controlled behaviors. So everything that I'll tell you about today is in the pathogen vibriocholera. So vibriocholera is a vibrio, so it's a quorum-sensing bacteria. And remember, I told you that these bacteria tinker with these components they use. So as far as we can tell, cholera only uses two of these circuits. And the way that it works in vibriocholera is that at low cell density, when the autoinducers aren't there, phosphate flows down through the circuit if egg it's made, that transcription factor. And it turns on biofilm formation, which is how bacteria sit on surfaces like on your intestinal cells. And it turns on the entire repertoire of virulence factors of poisons that cholera makes. So at low cell density, cholera is making a biofilm and it's a pathogen. At high cell density, when the autoinducers kick in and the switch gets flipped, half-R gets made and it turns off all of the canonical virulence and biofilm genes, but it turns on a protease that cuts cholera off the intestinal epithelial cells and out it comes back into the environment. So cholera has to have quorum-sensing to be a pathogen, but it's got this really insidious way that it uses this circuit. It sort of comes in, guns loaded. It's got all these virulence factors on. It makes a biofilm. You know, you get it from drinking contaminated water. So it comes in, it's highly virulent, and it gives you this terrible diarrhea. But then as the bacteria is growing and the autoinducers are accumulating in the intestine, when it flips its switch, it actually disseminates by the gazillions to infect the next patient. So in fact, it's all about dissemination. It causes this acute disease that if you survive the acute phase, you're actually cured because cholera is trying to get into the next host. So you have to have quorum-sensing, but quorum-sensing shuts down virulence. And so that was interesting for us because everybody, lots of people in my field want to interfere with these quorum-sensing systems. And in this case, the bacteria had done it for us. What we could show is that indeed, if we just added the auto inducer, we could shut down virulence. And so we could show that in a test tube. We could show that in a mouse. And we thought, that's pretty interesting because it gave a proof of principle that you could with a molecule interfere with virulence, right? And not an antibiotic, a behavior modification molecule. And so that was kind of nice because evolution had given us that. But evolution gave us kind of wimpy molecules. They're good for auto-inducers, but they're not really medicines. And so we wanted to understand whether or not we could expand this repertoire of molecules that we had to work with. First, to make probes, to understand how the system works. And second, to try to think about whether there really could be merit to this idea of deliberately interfering with these systems, right? In order to possibly make applications. And so when we decided to do that, that was happened to be at a, we wanted to do a screen for molecules that would be pro-chorm-sensing molecules, right? Pro-chorm-sensing molecules that would flip cholera into this avirulent dispersal mode. And why that is coming up today is because the person who did that is sitting right there in the front row. That is Amanda Hurley, who is currently Joe Handelsman's postdoc, and that'll tell you why I'm telling this story today. Anyway, she's not in tomorrow's talk. She gets one talk out of me. Anyway, so this fantastic, yeah, luckily for you, this fantastic graduate, then graduate student, Amanda Hurley came to the lab and she wanted to take this on as her project. And so what she did was to just get her hands on one of these libraries of synthetic molecule and screen for molecules that would turn cholera into this high cell density avirulent mode. And so sure enough, she got agonists of these receptors and they looked nothing like the auto-inducers and she and other members of the group studied them and we've characterized those molecules. But I wanna tell you about a different molecule that Amanda got. What she got was a molecule that worked on this protein luxo. And why we were particularly interested in that is because nature had given us molecules that work on the receptors, but we had never had a small molecule that worked from the inside of the cell. And so we thought this would be an interesting molecule to think about manipulating this protein luxo. And so this molecule, which we call ASAU, there's the structure of the molecule, is an inhibitor of luxo. So if you inhibit luxo, cholera flips into this high cell density avirulent mode. And so we were pretty interested in that molecule and we wanted to understand how does this chemical inhibit that protein? And in order for me to tell you about that, first I have to tell you about this central regulator that runs the show, luxo. So luxo is what's called a AAA plus ATPase. And this is a super family of proteins that exists from bacteria to humans. And so a famous one that you know about is called dimene, right? And so these are many proteins and they all are built sort of the same way. They detect something, they're regulated by something, and then they hydrolyze ATP to get energy and then they do a job. And so in the case of luxo, the way it works is that it is phosphorylated by those receptors, right? That's the regulation. And then that turns it into an active ATPase which gives it the energy to melt the DNA, right? To allow transcription to happen to be in this virulent mode. And then at high cell density, so that's at low cell density, high cell density, it's defosphorylated, the ATPase is not functioning so it can't open transcription and then these cells are not virulent and it's in the inactive form, right? So that's how the protein works normally and Amanda's molecule somehow converts luxo from this virulent form, active form, to this a virulent inactive form. And so we wanted to understand how does it do that? And so the problem for us was we didn't understand how phosphorylation does that. We didn't understand how the protein was normally regulated by quorum sensing, just naturally regulated and then we wanted to layer on top of that, well how is it synthetically regulated? So Amanda had to first figure out the natural form of regulation and then the synthetic form. And so what she decided to do was to do a structural study and so she teamed up with our colleague Fred Hueson and they made crystal structures of the molecule three times. The APO form, the ATP bound form and then the AZU bound form. And by solving all of those crystal structures they could figure out both the natural and the synthetic regulation of luxo. Okay, so here's one of the crystal structures. It crystallizes as a hexamer and that is very common for these AAA plus ATPases. They often make oligomers. And so now I'm gonna zoom in onto one of the monomers and I'm gonna show it to you three times. Okay, so first the APO form. So what you see is that this is the regulatory domain and I highlighted here in pink. This is where it would be phosphorylated by the receptors. So that's the regulatory domain that interacts with the receptors. And this is the catalytic domain that hydrolyzes ATP and just to tell you this is where ATP binds. So that's the catalytic site. So regulatory domain is next to the catalytic site and what was really interesting, right? There's lots of crystal structures of these AAA plus ATPases. What was really interesting is that the green looks normal, the blue looks normal but this little yellow linker which links the regulatory domain to the catalytic domain it's sitting like a plug, like a thumb right in the ATP binding site and that had never been seen before. So it looks like intramolecular inhibition. So when the protein is off, this thumb is sitting right there and presumably ATP can't bind. And so Amanda got evidence for that by crystallizing it bound to ATP. And so now what you're looking at here's the phosphorylated form of the regulatory domain. ATP is in the active site and so what happens is that little yellow linker just unfurls when the protein gets phosphorylated in order to let ATP into that site. So now the protein is active and so these two crystal structures told her how the protein is naturally regulated by phosphorylation. One of two things can sit in that binding site either the little linker and the protein is off or ATP and the protein is active. And then of course she has this synthetic molecule and what she found was that her inhibitor sits right in the ATP binding site. So in fact three things can sit in that site. The yellow linker thumb or the inhibitor molecule and the protein is inactive. If ATP is there, the protein is active. And so now I've told you both how the protein works naturally and how Amanda's molecule works and the reason that I'm going on and on about this little yellow linker is because usually AAA plus ATPs they just make different number oligomers. That's what activates or inactivates them. This business of having this little finger sticking in there in the ATP binding site that seems to be unique to Luxo. It's never been seen before and I'm gonna come back to that in a second. Okay, so now I'll go back to the circuit. So now I've shown you that this molecule, AZU slots into the ATP binding site to inhibit this protein Luxo. And if that's really true based on Amanda's biochemistry then that should make cholera a virulent. So then it was sort of like, put your money where your mouth is, right? Add this to a real cell and can we shut down virulence with a synthetic molecule? Okay, so this is the experiment that we did. So you're looking at a Western blot here and this is a protein called TCPA. That is the cholera, the pillace. When cholera gets into the intestine it makes a biofilm on the intestinal cells and it attaches with this pillace. So it's one of the first virulence factors that gets deployed when cholera enters the host. And so what you can see is that at low cell density it makes lots of that virulence factor. But if we add Amanda's molecule what you can see is that she can completely shut down the production of this virulence factor. And we can do that with any of the known cholera virulence factors, this molecule AZU suppresses them. And so I know that I'm either in or next door to a biochemistry building and so if you're actually looking at these concentrations it's ridiculous, right? So this is not a medicine, right? But of course what we wanted to do it is to show that we could actually manipulate virulence with a synthetic molecule. And this is the experiment that gave us some confidence that this approach might be reasonable. And so now what we're doing, so she fled and came here and left us with this mess to clean up which is to actually now do the really hard, no offense. I am offended to do the medicinal chemistry to actually change this from this proof of concept result to actually making this molecule much more potent, right? In order to actually see if you could move forward thinking about this direction. And not to damage Amanda's reputation much more. I'll tell you why I'm really interested in this. That is when you step back and you stop thinking just about cholera but you think about vibrios. So everybody's heard about cholera because it's famous and it causes this terrible disease, right? There are many virulent vibrios on this earth. They just don't get as much press, right? And so all of these virulent vibrios and there's many others have quorum sensing and in every case quorum sensing controls the virulence program. And these vibrios cholera included kill lots of people on earth every year and they mostly kill children. And so you cannot vaccinate these people. And so what is interesting about, if there's something interesting about these diseases is what I just told you is that they're all controlled by quorum sensing. So I've already told you that at the top of these circuits these bacteria tinker with these blends of molecules because it means something about self and other, right? So we and lots of other people are trying to tinker with these auto-inducers but doing those kinds of manipulation tends to be very species specific. Likewise, lots of people have a pony in the race of trying to ask can you interfere with the particular virulence factor to shut down disease? And so they're working on the bottom of these cascades. That's a profitable direction. But again, each one of these guys deploys different virulence factors and that's why you get a different disease. So what's interesting to me is what's in the middle. All vibrios have this intact. Every vibrio has Lux-O and nobody but vibrios have Lux-O, right? So I just told you that Lux-O has this special little way of inhibiting itself. And so sure enough, Amanda tried her molecule AZU on all of these pathogens and many others. And in every case, she can shut down virulence, right? But remember, there's lots of AAA plus ATPases and people don't like to work on AAA plus ATPases for inhibition because you tend to get off target effects because it's a super family of proteins. But none of them apparently have the little yellow linker. They work by these oligomerization states. And so in fact, we've tried this molecule on all kinds of other bacterial and eukaryotic AAA plus ATPases and it doesn't inhibit them. So on the one hand, the molecule is very broad. It's an anti-vibrio virulence molecule. But on the other hand, the molecule is exquisitely specific. It seems to hit Lux-O and has no target effects. And that is why even if she escaped, we are gonna keep working on this, right? Because I think there might be some way to really think globally about these terrible vibrios and how to manipulate quorum sensing by going after this Lux-O. And so that's sort of the highlight of that story. It was beautiful work that Amanda did while she was in the lab, right? And the fact that she got this molecule that worked from the inside both helped us to understand quorum sensing better and then hopefully eventually do something that's more practical for medicine or therapies. Okay, so that is my story. That's it, Amanda. So that is my story. She's been gone for six months so I need to tell you a newer story. Okay, so that, oh, I see. Is this a medical school so no one laughs at the jokes? Is that, I can't, I thought I was, okay, anyway. All right, I see how it's gonna go. Okay, so anyway, that's my story about molecules and the circuit. And now I wanna tell you a story about behaviors, right? Like what does it mean to do quorum sensing if you're a bacterium? Okay, so the behavior that I'm gonna tell you about is called biofilm formation. And maybe you've heard of it. So what we have learned in the last decade or so is it turns out that bacteria don't live shaking around in flasks, right? They actually live everywhere in the world on surfaces. So in an outside biotic and abiotic surface, we think that every surface in the world that is open to the environment is covered by an invisible or visible film of bacteria. And we call these biofilms, right? So this is the environmentally, ecologically, and medical way that bacteria live. And of course, if you have heard about biofilms in medicine, you've heard about them being the source of all of these recurrent infections. They are tenacious, they are hard to get rid of because they are adhered to surfaces typically in or on the body, right? And something happens to bacteria in biofilms. If bacteria are swimming around, you can kill them with antibiotics. The isogenic, exactly same bacteria, if they're in a biofilm, they are a thousand times more resistant to antibiotics. So something happens in how they change their behaviors when they're sitting in these communities versus when they're swimming around free, right? And quorum sensing, why am I talking about this? Because quorum sensing controls biofilm formation in many bacteria that have been studied, including in cholera, so we are intrinsically interested in that. And so what we know about biofilm formation, we have a cartoon from the field, is the bacteria that are motile, so they'll have a flagella, they will swim down and they will adhere to the surface, right, and then they will start to form a biofilm. So on this surface, they will change their gene expression and they start to make a matrix that is made of polysaccharides of DNA and proteins. And that matrix covers them like a suit of armor and it glues them together and it glues them down to the surface. And so they start to grow into these microcolonies and then inside this matrix, they will grow into these large three-dimensional communities of bacteria that we call the biofilm. And then at some point, some bacteria inside of this will decide that they don't wanna be in there anymore and so they will change their gene expression again, they will regrow their flagella, they'll make some hydrolyzes and bust out, presumably to find new real estate and start this over and over and over again. So that is biofilm formation. All of these transitions are controlled by quorum sensing and when I look at this, besides wanting to understand how quorum sensing controls it, what's interesting too about it is when the bacteria sit on the surface, they are necessarily in two dimensions, right? They're on the surface. But all the goodies, all the stuff they need to grow and make the mature biofilm is flowing by in the third dimension. So what I also wonder is, how can bacteria be two-dimensional and three-dimensional at the same time, right? And so we would like to understand just how it happens and the role of quorum sensing and so what's the problem, right? There are thousands and thousands of papers on biofilms. Well, the problem is that they are opaque and so in fact, no one has ever seen beyond this first step. No one has ever peered into a living and growing biofilm to ask what the cells are doing, how are they arranged? We don't have a clue. Who is it that turns on their flagella? Why are they a thousand times more resistant to antibiotics? Nobody's been able to see inside of these to actually even ask how the cells are arranged, right? And so we wanna understand it because of this quorum sensing connection and so at the time when we were starting to get interested in this, a physicist named Jing Yan came and he joined the lab and he said to me, oh, Bonnie's a physicist. Oh, Bonnie, this is not a biology problem. This is an optics problem. I can solve this. I mean, he's a physicist, right? So that's how they talk. And so anyway, so what he decided to do was to actually, so he built a custom confocal microscope in which, so the problem is that, why isn't anybody seeing? The problem is bacteria are tiny. I'm telling this to a micro department, right? But they are 500 or a thousand times smaller than eukaryotic cells, right? So there's a terrible phototoxicity problem when you try to image these and it's hard to segment them. So what Jing decided to do was to build this custom confocal microscope in which he sort of tweaked every single button to be imaging tiny objects. He reduced the pinhole size of the machine so they wouldn't get as much zap with the light and he built very bright reporters. And so he put all of that together and then the real genius was he wrote custom algorithms to segment the cells. And by doing all of that together, in fact, he cracked it. And for the first time, he could see in and watch a living growing biofilm form from the founder cell. So I'll show you his data. Okay, so cholera makes a biofilm from one mother cell. There's a founder cell, so everybody in the biofilm is a descendant. Okay, so now you're looking at the bottom slice of the confocal microscope. There's the founder cell, right? And then I'm just gonna turn it on, right? And what you're gonna see is that the cells divide and they spread out on the surface and they make this very ordered pancake-like structure. And what you have to notice is the cells in the middle look like points and that's because they've reoriented. And I'll tell you about that, right? You're looking down on it. So he does that and so then this is confocal, right? So he takes about 150 slices like this over time and then he stitches them all together, right? And so here's just some time slices from the, again, the bottom, the base of the biofilm, right? That's the pancake, but he's made 150 of those. And so here you're looking down on the biofilm. Now you're looking at it from the side, right? This is that hemisphere, like in my cartoon. So the colors go from low to high. This is about 21 microns high. There's 15,000 cells in it. So obviously what you can see is every cell that's on the periphery of the biofilm. But what I can tell you is where every cell on the inside is. I can tell you how close or how far it is from its neighbor. I can tell you how it's oriented and I can also tell you who its mother was, who its grandmother was and who its great-grandmother was, right? So he got the whole shooting match, at least at first blush, on how these biofilms form from the founder at least to these 15,000 cells. So now we can put some steps into that model. We can draw a better cartoon. Okay, so here's our new model. So indeed the bacteria swim down using their flagella. And color is a rod, right? So it's longer in one dimension, right? And so they sit down on the surface and they always sit on their long axis the way I have drawn in Jing's cartoon. And then they start to divide. They start to make this matrix that I told you about and that glues them to the surface and to one another and then they keep dividing. And now it really is about physics, right? So what's happening? They are glued to the surface. But every time a mother's cell divides in two, she has to make room for her daughter, right? So think about this as a disc, right? And so they're glued to the surface, but every time you make room for the next cell, there's this force pushing against you, right? So there's two forces. One that's holding the cells to the surface and one that's pushing from the middle outward against the cells as the cells are growing. And so at a certain point, the force of pushing the cells, my neighbors, away from me supersedes the force holding the cells to the surface. So the cells right in the middle of the biofilm, right? And that's where the force is the strongest, right? Because they have neighbors all around. In unison, the cells in the middle of the biofilm pop up onto their fanies and now it's over. They are in two dimensions because they are attached to the surface, but they're in three dimensions because they've reoriented. And so now every daughter cell that gets born gets pushed out further into the third dimension, right? Every time cholera divides, they just keep growing in the third dimension, right? And so then eventually you get the real thing that I showed you, the real biofilm that I showed you had 15 layers in it, right? My little cartoon here has just four, right? But it's over. The second this reorientation event happens, it's done and then they just keep getting bigger. And what is interesting, when you look inside these biofilms, they are not willy-nilly. They are lined up like soldiers. They are pneumatically ordered and there's less than one cell distance between each of these cells. And then they're sort of decorated by these radial cells around the edge. Okay, so that's what happens, but of course he's a physicist and I'm a geneticist and so I said, how does that happen? Or you know, and so we want to make some mutants, right? To understand the mechanism underlying that process. And so another thing you guys like back to here, another thing that's really great about this matrix, right, these are not eukaryotic cells. This matrix has exactly three proteins in it. So you only had to make three mutants, right? And what was even better, because he is a physicist, two of them proteins are redundant. So we only need to make two mutants, right? And so I'm gonna show you those two mutant phenotypes, right? So there's two proteins, you don't have to worry about the names, I have these colored. There's two proteins that make the adhesin that attach the bacteria to the surface, right? So he made a double mutant that can't make that adhesin. So what happens? Okay, so here's gonna be the movies. And again, you're looking at the base of the confocal, right? And what you're gonna see is it doesn't look like the wild type. They're kind of disorganized, right? They're not gonna make that nice disc, right? They're held together. Remember, there's still another protein and there's polysaccharide in there, but they're really disorganized, okay? And so if we stack them all up through the confocal, and I won't bother showing you, I'll just show you the cartoons, right? Here's this wild type that makes that nice hemisphere. So the mutant that can't adhere to the surface makes a sphere. And so it's just sort of sitting there teetering on the surface, right? And so of course, if you're not attached to the surface, you are not a biofilm, right? And so they're still adhered together. They're still, you know, in my logic, they're still in three dimensions. So they're growing because they're getting access to all this food. And so the evolutionary question is, well, what's wrong? Why is this bad when that's good, presumably, because evolution built it that way? And of course, there can be many answers for that, but we thought of one which has to do with the tenacity of these biofilms. Remember, they're adhered to these surfaces. You can't get them off, right? And so we started to think about perturbations, right? So we do all of this with our friend Howard Stone, who's a fluid flowed dynamicist. Anyway, he's a, anyway, he builds microfluidic things. So we do all this anyway in microfluidics. And so what we wondered is if we just added flow, what would happen? And so of course, if we add flow to the wild type biofilm, nothing happens. It sits right there. It doesn't care about flow. But in fact, this guy, even gentle flow, it's totally vulnerable, right? And so there can be many more reasons why they want to be adhered to the surface, but one is simply staying power. Okay, so that's two proteins. There's one more protein involved. So this green protein, what it does is it adheres the sister cells together. Okay, so Jing did the same trick. He asked what happens if you can now, they're gonna make the adhesion. They're gonna stick themselves to the surface. But what happens if the sister cells aren't connected? Okay, so again, I'm just showing you the bottom slice from the founder. And what you're gonna see is they look pretty good, right? And so here they're making that pancake-like structure. And if you remember the very first film, what you're gonna see is it's much bigger, right? And that's because the inter-sister distance is further apart, but they look pretty good, right? And so then we can stack them all up. And this time I really will show it to you. So now what I've done is it's turned on the side, right? And so here's the hemisphere of the wild type, right? See how close all the sister cells are together, right? And they're all lined up. Here's the mutant. It makes an enormous biofilm. And that's because there's a polysaccharide in there that is constrained by that protein holding the sisters together. So when that protein's gone, the polysaccharide expands and they make an enormous, but a less densely packed biofilm, okay? So that's the result. And so now I'll make it in a cartoon, right? The wild type is very densely packed, right? And then the mutant that doesn't have sister cell connections is bigger and it's got a lot of space in between the cells. Okay, so same question. What's wrong with this, right? Here you are taking a lot of 3D space. You have access to a lot of nutrients. Why is this a bad solution? Again, there can be many answers, but we thought of one that I'll show you today. And that I think has to do, now it really has to do with quorum sensing. So remember, these biofilms are made from a mother cell, from a founder, right? And when quorum sensing kicks in, they're making all these public goods. Hundreds of genes are being controlled by quorum sensing. They're making all these public goods that they're sharing in all of these resources, right? And they're all clones, right? So we think this is about invasion. We think you want to be really close to your sisters so that you're not vulnerable to cheaters. And so Jing tried to do an experiment to test that idea. So what we did was we added isogenic competitor cells and we asked, can they get in? So we just added swimming cells, right? And what you're gonna see with the wild type, they cannot get in. And so you have to forgive the colors are different here. So the resident biofilm is red. We have them with like RFP or something in there, right? So here you're looking down. Here you're looking, right? That's the hemisphere on its side. And the invaders are yellow. And what you can see is that they can kind of, they decorate the edges. They kind of get in little grooves, but they actually can't penetrate the biofilm. It's impervious to them. Okay, so what about the mutant? We add the isogenic cells and you're gonna see they went their way in and they can cheat. Okay, so now the resident biofilm is blue. Looking down, here's from the side. And what you can see is that those yellow cells can get in and in fact, they can get all the way to the surface, right? So we think then that this packing is like the tight junctions in eukaryotic cells, right? That they need to have less than one cell distance so that no invaders can sneak in there and take all of these goodies, right? And we have absolutely shown in more fancy competition experiments that these guys get outcompeted and these guys do not, at least the way we're doing the experiment. So there can be many more reasons why they have those three proteins, but we think that those at least are the beginnings of why nature might have evolved those proteins. Okay, so this is the state of affairs. We have this technology now. And so now the question is, what else do you do with it? And so I'll just tell you the experiment, the Drew Bridges, a current postdoc in the lab is doing is to actually ask about quorum sensing, right? So now that we can see at single cell resolution, right? These biofilms, as they mature, we can finally ask the question of who's doing what, when, and where? So we can put space and time into this. And so an easy experiment that we're doing right now is we've simply added colored reporters to our two favorite master regulators, AFA and HAPR, right? And then we can just ask, who turns it on, when, and where? And then you can get more fancy and do lots of colors and do it genomically, make mutants, add auto-inducers and ask using these kinds of technology. Finally, we can get a spatial temporal map of any gene you might like. Ours are gonna be the quorum sensing genes, but what is the program gene expression in space and time in these communities? And so that is sort of, not sort of, that is what we are trying to do right now with this, now that we have this microscope. Okay, so to wrap, finish up, let me go back to my circuit that I've told you about. So I've told you some of the strategies we use, both to find components and then figure out about how these communication circuits get deployed when the bacteria are being in communities. And of course I think that by finding these things out and having these pros, we can both learn about quorum sensing and just how multicellularity evolved on this earth, but also the hope is to do something useful in the future by actually, for good or for bad, manipulating pro and con these different quorum sensing systems. But then one more thing, besides this space and time aspect of this, that we'd really like to understand if we're really gonna learn about quorum sensing and if we're ever gonna make a medicine or an application, it doesn't get deployed in a test tube. It's gonna get deployed with bacteria in a biofilm, but those bacteria are very likely gonna be in the microbiome. And so I alluded to this at the beginning of my seminar, right? We all live in these consortia with these microbiome. Cholera, this bacterium I'm telling you about is an intestinal pathogen. So when it comes in to a human and quorum sensing starts happening, that is in the context of 10 to the 14th other bugs, right? So there are, at least at last count, people think there are about 600 different species of microbiome bacteria in your gut, right? And so somehow cholera's gotta come in and not get confused and use quorum sensing to be a pathogen, right? And so we know from sequencing and sampling studies that these microbiome bacteria make buckets of at least the auto-inducers we know about. So how is this conversation really happening? And so of course the answer is we don't know, but Justin Silpe, who is a current graduate student and Kai Papenfurt, who was a postdoc and is now a professor in Germany, they wanted to ask, knowing what we know, could we like start to think about mechanism here? And so Justin found our newest quorum sensing auto-inducer. It's this molecule, did Helen have to leave? Maybe, oh, there, she's still here, okay? So this is our newest molecule, we call it DPO. And what's interesting about this auto-inducer is it's made by the microbiome. So the microbiome bacteria make that molecule called DPO and then cholera has a receptor. It's a transcription factor, so it's in the cytoplasm and this molecule gets across the membrane and it's bound by this transcription factor and binding of the auto-inducer activates the protein to bind DNA. And so what you learned from me is that auto-inducers and cholera mean don't make a biofilm. It means turn on quorum sensing and turn off biofilm formation. That's what I've been harping on for this entire lecture. So what about this new molecule that Justin found? Okay, so we have an easy way to do this on a Petri plate. So this is cholera and these wrinkles, that's a biofilm. And so if Justin adds DPO, so once he figured out the molecule, we synthesized it. So if we add DPO, cholera shuts down biofilms. That should make sense with everything I've been telling you about. So no DPO, biofilms are on, plus DPO, biofilms are off. Well, what about quorum sensing? So quorum sensing is the reverse. So we just have an RFP, Justin made an RFP fusion to a favorite quorum sensing gene. So what you see is no auto-inducer, no quorum sensing plus auto-inducer, quorum sensing is on, right? So biofilms off, quorum sensing on. That should track with everything that I've told you about so far today. So then Justin figured out how this molecule is made. And so it turns out that DPO is made from two amino acids, from threonine and alanine, and then there's two steps with a tautomerization and blah, blah, blah, and then it gets cyclized. We actually know the steps, right? But it's, sorry Helen, it's boring. Anyway, so, and then, no, I mean, I think, anyway. And so anyway, but then by these steps that Justin figured out, you make this molecule DPO. And I'm like, are you kidding me, threonine? I mean, are you kidding? Like this is not a threonine? I'm like, where's all this threonine coming from? And that is when Justin told me, right, that it turns out that these microbiome cells live on just a little way away from your intestinal epithelial cells. So your intestinal epithelial cells are covered with a protein that's called mucin and it's like no man's land. It makes this little 50 micron region and the microbiome cells sit on top of it, right? And they eat mucin, they have mucinases. And it turns out that mucin is 35% threonine. And I'm like, of course, threonine was always my favorite amino acid, right? And so anyway, so it turns out that this I think is a three way conversation where the host, the human host is making the substrate, mucin. The microbiome cells are converting the substrate into this auto inducer, right, DPO. And then the pathogen is detecting that auto inducer and what you heard from me is when cholera at least detects an auto inducer, it disperses. And sure enough, Jeff Gordon's lab showed that if you have microbiome cells that have this enzyme and cholera has this receptor, so you have to have both parts, if you're a mouse, you get less cholera disease. So I think this is the first inter kingdom three way conversation where the host and the microbiome are teaming up to fend off pathogens. And at least in this particular instance, it's through quorum sensing. And so I think that there's gonna be, I've, of course, in brainwash, I think this is a treasure trove of molecules, but I think we actually know enough about the components of quorum sensing and we are starting to know enough about how the microbiome works to actually bring mechanistic insight into this. And actually, I assume that there's many more sort of different instances of things like this where these cells are actually communicating through these small molecules and whether it's a beneficial conversation or in this case, it's a hostile conversation, right? That's gonna matter, whether it's health and disease. And then, of course, I also think that the human host is playing a part in that. Okay, so that's our newest story and I will end with that because I think you can see where we're going. We're doing this space and time in biofilms and we're going there to find more molecules. And so that's sort of the next, I think, couple years for the lab. But let me just finish with the heroes of today's story. So of course, the big one is Amanda Hurley and again, you guys, I had the very good luck and the good fortune that she joined my lab as a graduate student and she did this beautiful thesis part of which I told you about today and now you have the good luck and the good fortune of having her around you and you will enjoy that and learn from her and she from you. So that's Amanda Hurley. She did the Luxo crystal story. Let's see, this is Kai Papenfurt. He's the one that found VQMA and figured out that new receptor in cholera. This is Justin Silpe. He's the one that figured out DPO. He's still a graduate student in the lab and this is Jing Yan. He's the physicist slash material scientist who built the microscope. And then again, we have these two wonderful collaborations with Howard Stone and with Fred Houston. Thank you for having me for part one. We'll see if the audience is empty tomorrow. But anyway, yeah, no, thank you for having me. It's a real delight to be here and to get to tell you our stories. Amanda Hurley is thinking to join the witness protection program. And now that you threw a drinking, what a special... It's water. Of course. What special genetic features endows a founder cell to be a founder cell and why don't all others break away and become their own founder cells instead of following the pack? Yeah, I think that... I don't know. I have... It's fantasy. I think it's mechanical. They sense mechanical that they've hit the surface and that induces them to start making those matrix proteins. And I think the reason they cannot... Lots of... If you sprinkle them down on petri plates, you get a gazillion of those biofilms, right? So every cell that hits the surface fast will grow into its own biofilm. But they are... They cannot be mixed. So Andy Camilli did this experiment if you just put red and green cholera in a mouse. They're isogenic except the colors. And you open the mouse up, you have a red biofilm, a green biofilm, a red biofilm, you do not have a yellow one. So cholera, it's clonal. And I think the second that they start covering them themselves with the matrix, whether you're a self or other, you can't get in. And they do break away. I think when they get big, somebody turns on those hydro lasers, they make their flagella and they do break away and go do it again. And we don't know what is the cue that tells them to do that. It could be something simple, like they run out of nutrients. It could be something simple. And so what we did, we can't see that step yet. So we bought a light sheet microscope because now we can look down on them because we really wanna see that dispersal step and ask who is making that decision. And we're also trying to make mutants that can't do it. And that's, I think that, this is a lot of words to say, I don't know. But cholera, it's clonal, right? They wanna give this stuff to their babies and nobody else. It could easily be. Absolutely. And a bee. Exactly, remember, so there's all these auto-inducers involved, right? And like if you looked at the molecules, right? This is fantasy, right? One of them is really hydrophobic. One is really hydrophilic. One is kind of in the middle. One has a long half-life, short half-life. It could be, right? That's even the molecules that we know about, right? That they have different diffusion inside the biofilm, right? And that that is telling, giving cells their fates. And it can be other molecules. I, of course, hope it's the quorum-sensing ones. But it can absolutely be that, right? That you have gradients like in a fly embryo, right? That you have these gradients or these molecules that are giving cells something like fate, you know, except that they can go start it over again, right? Yeah, I think, and I think that having that imaging, we're gonna finally, there's a fantasy, be able to actually start asking that question, which has been inaccessible to us before Jing did that trick. Yeah. So with regards to your very, the very last part with VQMA and DPO. So basically what the model that you proposed was that DPO gets in, binds to VQPA. Close, yeah. I know which one. That one, that one. Yeah, that one. I know where to go. I hate when people do that to, whoops, there. But yeah, VQMA, yeah. Yep, and so then basically it turns off biofilm formation of virulence. So if, and if that's the case and the bacteria in your gut are making the quantities of this, how then is it possible that Vibrio ever makes a biofilm at all if you've got 10 to the 14th bacteria making this deterrent? Yeah, it's a good question and it can be. So not all the microbiome, this Jeff Gordon's work, not all of them have mucinases, right? So it could be if you are unlucky, I don't know this, just I'm making up. It could be that the people who are more susceptible, you get less cholera disease. You don't get no cholera disease with the one I just showed you. But it could be that depending on your blend of microbiome cells, the people who are more susceptible to cholera are the ones that have microbiome cells that have fewer or less mucinase activity, right? So you could do this both ways. If you put it, he did a simple term where you put a bacterium in that didn't have the mucinase and it didn't matter, right? So that's just a fit that I, you know, I have no data on that, but you can think about that. And so then I'm thinking we can just feed people three anine, right? I don't think that's crazy. Yeah, Alanine is better. Alanine is better, right? Yeah, yeah, it's cheaper. Yeah, yeah, yeah. I think you might be able to do this, something like that, right? Yeah. So do archaea or eukaryotic microbes quorum sense? And is there any evidence for inter-kingdom communication in microbes? So those are two questions. So the first one is there are tantalizing hints about yeast, which I think is a good way to step your way up to humans. This molecule tyrosol, Jerry Fink found this, that he thinks that might be a quorum sensing molecule that tells yeast to differentiate between what do they call like a cell in the hyphae, right? So there's some evidence. They also grow on it, so it's a little bit tricky. You know, like ours, they don't, you know, you can separate easily growth from behavior, right? So there's a little murkiness there, but that's sort of a hint. No one's ever found this in an archaea yet. And again, I think that, right, so those are the hints. People are starting to talk about this in immunology, right, that these, that immune cells count themselves with molecules. And so I think, you know, a bonafide quorum sensing molecule in anything but a bacterium, not yet. And then again, what I can tell you, and that's why I put that one, it's not those. You know, so we have the genes that make the enzymes that make those molecules, and they are not in eukaryotes, right? And so, but then again, I always think for us, if it didn't turn on bioluminescence, just looks like water, right? You know, and we never put them in mixes. We haven't put them, you know, and so that's why we're trying to do the things that I'm telling you at the end. I think that there's this invisible set of molecules, right? Out there, for sure in bacteria and maybe in eukaryotes. You can imagine if you wanted to eat a bacterium in your eukaryotic microbe, you might like to chase down their quorum sensing molecules. You might have receptors for those. Right, so, or you might make mimics. So we have a molecule now. We don't have the structure of it quite yet, so it's a little faster prime time, right? So this molecule, this interspecies one, that is the most broad spread. It's like a molecule. There's buckets of that in the intestine. We know that human intestinal cells make a mimic of that and it's detected by this receptor. And so I think they're dry, like if I were a eukaryote, right, and I was gonna try to manipulate this conversation, I'd go for that molecule because it's the generic one. And so it looks like we don't have the structure of it yet. We almost do. It looks, it's six carbons with a thing on it, which we don't know what the thing is yet, but it's pretty close to the same structure of this. And we have a yeast mutant that doesn't make it. We have an enzyme in yeast that doesn't make it. So we know human cells make it. We got the mutant in yeast, right, for obvious reasons. And anyway, and so I think that they are driving these conversations, right? And we can only look for molecules that impinge on our receptors, right? There could be all these others, right? You know, like the, yeah. So anyway, but I think that this business of this being an inter-kingdom dialogue, that is on the horizon. Like, you know, the DPO story is, because the microbes are doing it, but I think that the eukaryotes, whether they detect them, and for sure they make molecules that control core sensing, I think that's common in the next couple of years, yeah. Hi. Hi, a very nice talk. So I have two questions. One is a kind of hypothetical question and another is a real time. So how the middle, once the biofilm is formed, how the middle layer cell is different from the cell which is at the periphery of the biofilm and how it is different from the mother cell in the phenotypic terms? And the second hypothetical question is if I take another cell, another cell from outside the biofilm and put it somewhere in the middle, will it be able to know that, yeah, it is now in the middle and how it has to behave? Right. So I don't, yeah, okay. So both in real life and hypothetically, I don't know the answer to this question. That is why, right. So now what we're trying to do, that experiment where we do with the colors, right? Where we actually ask, what is the gene expression program of every cell inside there in time and space? And so I showed you, you know, with a color, but we have a genomic way to do it, right? And that is the question. Are they like cell fates? Like I'm the car mechanic, I'm the grocer, I'm the librarian, right? And so that is what Drude is trying to do right now because we can actually see them and so we can see them either with RNA-seq, we can see them or with colors. We can actually see individual cells, right? And like, remember, these are small, so we've never been able to like just take, that'd be fun, one cell and put it in the middle, right? But I think what we can do with things like optogenetics and things like that, we can make a cell change its fate in the middle, you know, that was already there and then ask what happens to it and what happens to its neighbors, right? And those are the kind, that is exactly the experiment, theoretically is happening while I'm here. They're not, they're out to dinner. No, but anyway, yeah, you're on it, right? That is what we have to figure out is who's doing what and when, right? And we don't have a clue, we don't even know for those yet, right? We don't even know who makes the adhesive, right? Is it just the bottom of cells or do they, you know, we just don't know, but that's what we're on the hunt for that, yeah. So while I was a graduate student, I became really interested in the phenomenon of reciprocal regulation and E. coli. So of course, just H and S and LRP sort of titrate these processes. So I'm curious, it's really my sort of depth of understanding this regulatory appality, but in Vibrio, are you able to sort of following up the previous question sort of thwart a lot of the quorum sensing phenomenon if you delete these nucleoid structuring factors or overexpress them? So I wonder if that might be part of, you know, tuning these. Yeah, so I did, yeah. I think yes, the answer is yes. So we know that it was H and S, that H and S is part of this. We know that, that it regulates lux O, but I did you the favor of not drawing the nine feedback loops into this circuit? So there's plenty, plenty of reciprocal like little feedback loops that I think allowed them, right? They change the dynamics fast and slow. They allow them like to pay attention to one auto inducer and not another, right? So there's all of these engineering feats built into this circuit, right? That are what you're saying. They're little knobs that allow them to change the timing, the strength, and which, you know, to pay attention or to ignore different signals, right? In addition to these nucleoid proteins, which I'm sure are also layered on top of that in a more general way, right? Yeah. So I really like the idea that the biofilms are kept keeping the Vibrio clonal. It is very cool. When you make that not possible with the mutants, are they pathogenic? Number one, and number two, are there other bacterial species that actually want to be symbiotic together and change the program so that they're working either in twos or threes? Right. So question one, we haven't done it, but I think that you're right. This is an Achilles heel. Like if you made them looser or whatever, you know, I think they would get invaded and they would be less pathogenic, right? We have not done it. You know, you need to do that in a mouse, right? But I think that's a good idea to do. Like that is a real target, right? Among these things, right? And then the second question is yes, that this is one strategy, which is clonal. Pseudomonas, which is a notorious biofilm former, it doesn't do it clonally. And right, so if you did this green-yellow, you'll have green-red, you have yellow, right? And so I think the different bacteria that are carrying out whatever billions of years of evolution, their biology dictates to them, have actually solved this in different ways. And then the other thing we've been thinking about, like what about like bacillus, right, that are spheres, right? How do they break that symmetry, right? It cannot be what I just told you, right? Whether it's clonal or non-clonal, right? Because they're circle, I mean, spheres, right? So I think that there's, so I think that Jing would like to study, you know, he's a physicist, so he thinks about these as materials. And so I think like actually stepping his way through a few of these other solutions to get the big principles out, I think would be a really good direction, you know? And so that's how, yeah. So that is the answer to your question. All right, in the interest of time, please take your questions offline at the reception. And let's join me in thanking Bonnie for a great talk.