University Place Wednesday Nite @ the Lab Ned Ruby, "How Bioluminescent Symbioses Benefit Animals" 9/29/2010  UP 491   cc >> Welcome everybody, to Wednesday Nite at the Lab. I'm Tom Zinnen. I work here at the University of Wisconsin Madison Biotechnology Center. I also work for UW Extension. And on behalf of the Wisconsin Alumni Association and Wisconsin Public Television, welcome to Wednesday Nite at the Lab. We do this every Wednesday night 50 times a year. Tonight I'm delighted to have Dr. Ned Ruby. He's the second part of our double-header on the beneficial interactions between squid and Vibrio bacteria. Dr. Ruby has been contributing to the understanding of the role of bacteria and bacterial host signaling for over 30 years. He got his doctorate at Scripps Institution of Oceanography at the University of California San Diego in the field of marine microbiology. 

He worked on luminescent bacteria isolated from the light-emitting organ of fish and helped discover the cell-cell signaling phenomenon that is now known as quorum sensing. He then received an NSF postdoc fellowship to work at Harvard focusing on the comparative enzymology of bacterial luciferases, and then he joined the Woods Hole Oceanographic Institution where he collected the first bacteria isolated from the newly discovered deep sea hydrothermal vents. He did a final postdoc at UCLA, and in 1982 he took a faculty position at biological sciences at the University of Southern California. In 1989 Dr. Ruby began a collaboration with a developmental biologist, Dr. Margaret McFall-Ngai, who was our speaker last week, to study the two sides of the animal bacteria symbiosis. Working together, they developed the exquisitely elegant squid Vibrio light organ association as an experimental model for the beneficial host microbe interactions. In '96 they were recruited to go to the University of Hawaii where the abundance indigenous host animal interactions facilitated their research and the use of genomic tools. There they laid a foundation for understanding the role of bacterial signaling in triggering the differentiation of host tissues during development. In 2001, Dr. McFall-Ngai and Dr. Ruby were awarded the Miescher-Ishida Prize for contributions to the field of symbiosis. And then in 2005 at the initiative of the provost here at UW Madison both McFall-Ngai and Dr. Ruby were hired into the symbiosis cluster as members of the Department of Medical Microbiology and Immunology. Please join me in welcoming Ned Ruby to Wednesday Nite at the Lab. 

(applause)  >> Thank you very much, Tom. Great introduction. And welcome to all of you. I'm very glad that you were able to come and join me tonight while I tell you about a subject that I've had a great deal of interest in over the years and that is bioluminescence. For those of you that were at Wednesday Nite last Wednesday night, you heard from Margaret McFall-Ngai, as we've just heard here, and what she has spoken about is the animal side of a conversation that we've been studying for a number of years. Now, a symbiosis between a light emitting bacterium and a squid may seem pretty exotic and pretty derived and may be only interesting to about five or six people on the planet, but in fact the reason that we study this not that we're interested in either luminous bacteria or in squid. It's because we're interested in how bacteria in general interact with their animal host. As I'm sure Margaret has told you, all of us sitting here are just vehicles or vessels for bacteria to get around from one place to another. And those bacteria are very important to us just as we're important to them. And understanding that symbiosis and the degree to which that's critical to our own health is an important part of understanding not only health but also disease. 

So what I would like to do today, though, is tell you a bit about how we've learned something of how bacteria and their hosts get together. And to do that I'm going to have to introduce you to the major reason why this particular symbiosis has evolved and that is because many animals want to have the ability to produce light. All of us in here want to have it whenever the lights go out at home, we want to find a flash light, we want to be able to see in the dark. And we're not alone. Animals like to do this as well, especially if they live their entire lives in a dark environment or spend their days asleep and their nights out in the environment. So what I'd like to do is go through this series of questions that I have up here. First, what kind of organisms make light and where are they found? And also, what is the way in which, what's the chemistry behind making biological light? We all understand how these lights work but there's organisms and cells that are able to make light just like the kind of light that we're seeing here being made electronically. And the third I'd like to talk about, the special link that exists between bioluminescence and symbiosis. Because as I mentioned before, the ability to produce light is a really high and strong fitness advantage in many organisms living in dark environments. And the ability to capture that capability from another organism, in particular a bacterium that you can raise within your own tissues, is a great advantage. 

And in fact, many, many organisms have done that, as we'll talk about. And finally, if we have time, I'd like to talk a little bit about a specific question that, as I say, is important not just to this particular symbiosis but is important to all symbioses that humans have and that is, how do we find and enter into relationships with our beneficial bacteria? And how can we tell the beneficial ones from the bad ones because obviously it's a big chance opening up your tissues to bacteria. There are bad ones out there. Actually not very many of them, but the ones that are there can cause us great deal of problem and disease. So this interacting with bacteria is a two-edged sword. And the key is to be able to pick your partners carefully. So I'd like to tell you a little bit about how we're learning about the process that these particular partners have used to create this symbiosis together, and hopefully that is going to tell us a little bit about how that is going on in every one of us when we initiate our symbioses with our microbiota. So first I'd like to start out talking about something that we all know about, anybody who's lived here in Wisconsin in the summer, knows about fireflies. And of course, the first bioluminescent organism, if I were to ask you what ones you know about, would probably have been the firefly. Some of our pictures here might not be too entirely clear, but what we've got is just a couple pictures of fireflies. 

These are actually from Indonesia. They are not the ones that you can find here in Wisconsin. Over here there's just a picture taken by its own light from underneath the animal. Once again, pretty much anybody who has caught a firefly has seen how they make light in their abdomen. But there are other organisms that produce bioluminescence that are found in the terrestrial world. Mushrooms, not all species by any means, but there are a few species of mushrooms and actually ones that you find here in the northern climates that produce bioluminescence. And they're actually quite an eerie glow if you run into them at night walking around. Not too many people walk around in the dark at night in the woods, luckily. But if you were, you'd have the opportunity to see these organisms in certain places. It's a little too sensitive. 

So there's also glow worms. And these are one of my favorite ones. There's apparently caves down in New Zealand that I've never gone to, I'd like to, that the ceilings and walls are covered with a glow worm. It's actually a fly but as a larva it produces, these worms are the larval form of the fly. And they produce these large webs and colonies as they're developing into the fly. In these deep caves, deep dark caves, and apparently at least at one point you can go in there and just look at it and it's like a big cathedral of light up above you. All bioluminescence. And down here, finally, there's these bioluminescent bacteria that some of you maybe able to see. And they're dots of blue light. And they're basically colonies of light emitting bacteria. 

These bacteria can be found in special light organs that are produced by a small nematode. And that nematode can be found in many places in the United States. And there are a few labs that are working on this particular symbiosis between Photorhabdus, this terrestrial luminous bacterium, and the nematode, the worm that they live in. But by far the place where most bioluminescent organisms are found is in the marine environment, and in particular in the deep sea, but also in the shallows of the sea, at least during the nighttime. And the reason for that is pretty clear. When you look at the ocean you can see that there's actually a number of different areas within the ocean that are designated by oceanographers. And the epipelagic area up there at the surface is the area that experiences day and night just like we do here in the terrestrial world. But once you start going deeper into the water, that light gets absorbed by the water. And once you've gotten down to the bathypelagic area, which is below about a thousand meters, not even the strongest light gets down there. And down in that environment there's no change in light from day to night. 

And so this world from a thousand meters all the way down to the deepest 10,000-meter depths of the ocean, there is no light except the light that is produced by organisms. And the interesting thing is to realize that there's a very large number of organisms down there that produce the light. And the first people to go down in bathyscaphes into this deep sea and look out through the windows were amazed at the amount of light that was down there, the number of organisms that produce light. So clearly the ability to produce light and use it in your behavior is something that's a key to these various organisms that are found in the deep sea. Now, as I mentioned, light is being attenuated, it's being absorbed as it goes down through the water. And I'm sure many of you who have gone into water, even into a swimming pool, will notice when you get down even as shallow as six feet, you will notice that you look around and everything you see is blue. The only light that you seem to be able to see is blue light. And that's because not only is light absorbed by water, but it's absorbed differentially depending on its wave length. So as we can see here, there is a difference between the degree to which red light is absorbed and blue light is absorbed. And, in fact, red light is very quickly absorbed within the first two or three meters of water. 

All the red light that's in the spectrum of white light that comes from the sun has been removed. And by the time you get down to 40 meters, the only light that's present there is blue light. So not surprising, and that's because blue light travels further in water than these other wavelengths of light. So not surprisingly most organisms in the deep sea produce blue light. There's no use producing a light that can only go a little bit in front of you. You want a good light. And that means a blue light. So most of the bioluminescence that you'll see in the ocean is a blue light and that's why. It's simply based on the physics of absorbing light in the sea. Now interestingly, and not too surprisingly to us I hope, the biological world responds to these physical constraints. 

And in fact, if we look at this graph here which is simply a way of looking at the number of the organisms that have photo pigments in their retinas, that is the pigments that collect light and tell us what's going on around us, based on what wavelengths of light that photo pigment is best able to see. And if we look in the terrestrial world, we see that most of the light that's easy to see is up around the yellow and green area. Sunlight is kind of a yellowish, dominated by yellow light. And so not surprisingly, most organisms are able to see that light best in the terrestrial world, most terrestrial organisms are. But as we go into the ocean, both coastal and especially into the deep and pelagic areas, we see that now the wave lengths of light that are best picked up by the pigments that are specifically present in these deep sea organisms are down in the blue region. And so the tracking of the ability of the light of the eye to see a different color of light is dependent on the environment in which the organism lives. And there's no use looking for red light if there's no red light in the environment that you live in. And so these eyes are set for blue light. So what kind of organisms in the marine environment produce light? An incredible array. Just about every aspect, every phylum, every group that you can imagine has some members who produce light, not all species, of course, but some members. 

And so here we just have a few. There's an octopus that, many cephalopods produce light, but here's an octopus that's producing little areas of light all along its arms. You can see the white dots there. Dinoflagellates that produce light. Any of you have been in Boston or around Massachusetts, Cape Cod, in the summer and have splashed around in the water have perhaps seen light coming up out of the water wherever you disturb it. Those are dinoflagellates. They're little small algae. They're not that big, I don't know why I did that. But they're very tiny algae. And those algae are all capable of producing light, and that light is actually initiated by mechanical simulation. 

And so it's very exciting to run around in water there and watch the splashes that you make produce light. Over here there's a little picture of a euphausiid shrimp. And they've actually put him on top of a mirror. And what you're seeing, those little blue spots down below are the blue light, the little blue headlights that he has all along his ventrum and is showing back up in the mirror. It's a very clever way to show this bioluminescence on the underside of an organism. Many organisms produce bioluminescence on their ventral region, and I'm sure Margaret spoke about this last week. But the reason that they produce light on their ventrum is because it's a part of a behavior called counterillumination. And this is often done by organisms that live not in the deep sea but up in the epipelagic region where there's a little bit of light that comes in every day from the daylight. Or perhaps if they're very shallow even at night from the moon. And the problem is if you're out at night, you don't want to be seen by a predator and you're swimming along in the water, if there's a light above you, whether it's a sun or a bright moon, above you, you'll cast a shadow. 

You'll cast a shadow down on the ground below you just like I'm doing here. And there are predators that will come along, well, they just sit there and they look until you come along and as soon as they see a silhouette up there, they shoot up, grab you and you're gone. So the key is not to make a shadow. So if you can produce the same amount of light on your ventrum, on your bottom part, that's the same level and same quantity as you're occluding by your silhouette, you effectively become invisible to the predators down below. And this is called counterillumination. And this counterillumination is a behavior that many, many organisms in the surfaces do, especially those that are out and active in the open ocean where there's not very many places to hide. So you've got to figure out how to make yourself invisible, and this is a great way to do it. So how about vertebrates? I've talked about a number of invertebrates there, but how about vertebrate organisms? Well these are difficult pictures to see, but what we've got on the upper left there is something called a lantern fish. And basically the lantern fishes have all along their sides rows of lights. And so they actually look like the lanterns that were hung out on the sides of old sailing vessels back in the day. 

And so they felt these fish looked like lanterns coming along on a boat. And those bioluminescent organs are used in a different behavior by this particular animal. There's also this hatchet fish which is probably one of the most abundant species in the deep sea that also produces a bioluminescence on its ventrum. In fact, you can see this long extension here. These are actually light tubes. The light's made in an internal organ and is shone out through these tubes down to the base of the animal. Now, everything I've told you about now has been something that is up there in red called autogenic luminescence. And what autogenic means is the organism makes it themselves. Self-generated luminescence. And fish, many species of fish, many species of invertebrates do this. 

But other species of invertebrates and fish have figured out that it's actually easier to grab another organism that produces light and incorporate it into their lives and have that be their flashlight. And just maintain those organisms and maintain control over those organisms and that way they have a light emitting system that's already pre-made for them, and they don't have to do the chemistry themselves like fireflies do. And so there are a number of fishes that have this capacity. I think many of you are familiar with anglerfishes. They're very ugly looking organisms. But they're very functionally good. They're basically a big mouth. And they have these lures that come out and they usually sit and wait for something to come and grab this seemingly food item here and then they grab them instead. And in these shallow waters that little lure that they're putting out there is just a wiggly little thing, looks like a bunch of worms. But in the dark, of course, there's no sense wiggling a bunch of dark worms. 

And so what you've got to do is make them light. And that's what this organism does, and it does that by putting luminescent organisms, bacteria in this case, out in those lures called --. And they're put out there and that light is being produced all the time and is allowing the animal to draw in its prey. The rattail down here is another deep sea fish. It's got a perianal light organ. That is, the light is being produced around the anus. Nobody has a clue what that's being produced for. Some of these behaviors are, oh my, some of these behaviors are very dramatic. Here's another shrimp, not a euphausiid, over there on the left. You can't quite see him. 

And what he's doing, actually, is facing off a fish there on the right, and just as that fish is coming to get him he's spitting out luminescence to blind the fish and that'll allow him to shoot off in another direction while the fish is thinking about what's going on with all these lights in front of me. And this is a very useful way to distract the predator that a number of organisms do. And so this is this distraction behavior that I've just described. I've also described counterillumination, that's another important and very common bioluminescent behavior. And I've mentioned the attraction of prey, which the anglerfish does. But I haven't talked about interspecies communication because luminescence can be a really great way to signal to other organisms. And there's an organism called the flashlight fish that's found in a number of places but mostly in the Red Sea. And they have little light organs down around their jaws. And they're able to cover that and sort of flash on and off in whatever pattern they want to. And there have been some great experiments done at Steinhart Aquarium in San Francisco, many of you I hope get a chance to go there, it's a wonderful aquarium, and what they've shown is that if they put two fish in a tank on either side of a divider, one will start making flashes that it doesn't make in the absence of another fish to look at. 

So it's believed that there's some communication going on there. What we're seeing here, I hope, is a black dragon fish, and the black dragon fish, you probably can't see the outline of him, but he's got a lure down there, that's a little blue lure down there, but he's also got a red light emitting organ up here and a green one right behind his eye there. And remember I told you not many organisms in the deep sea have the ability to see red light, but if you look at this graph that I showed you earlier, there are a few organisms that have the ability, have pigments, photo pigments in their eye that let them see not only blue light but also red light. Well, what these dragon fish do is they produce a red light which they know no one else is going to be able to see except another member of their species. So this is their way of having a secret code that they can communicate with amongst the species that nobody else can see. So it's sort of like having infrared glasses. You can see what's going on and nobody else can. And so that red light production, although it's rare, has been re-evolved as a way to have a specific communication. So now I'd like to spend just a few minutes telling you a bit about the chemistry of the bioluminescence. How do you make light? How complex is it? Is it really that difficult? And how many times did it evolve as a chemical capacity? Well, basically, it turns out that the ability to produce light is relatively simple, chemically speaking. 

It really only takes three, sometimes four, factors. One is an enzyme. And that enzyme has the generic name luciferase. Now there are many, many different luciferases. Every species of animal has its own distinct individually evolved luciferase. But it's a protein that catalyzes a reaction that produces light. That's all luciferase means. But luciferases always have a compound that they're oxidizing. And during the process of that oxidation, light is produced. And that compound is called luciferin. 

And luciferin goes from a reduced state in the presence of oxygen and luciferase to an oxygenated state. And during that process there's an excited state produced in one of the chemicals that then gives off light as it decays. And that light then is bioluminescence. Now sometimes ATP is necessary, in fact our friend the firefly has to use ATP in this reaction to produce light. But organisms also need to use energy to convert that oxyluciferin, that oxidized luciferin back into the reduced form or reduced chemical form so it can go through another cycle because this luciferin is being oxidized and reduced over and over and over again producing a photon of light every time it goes through that reaction. That reductive recycling process requires energy. And that's the energy input that is eventually gotten out by the production of light. So that's what you're giving up in order to be able to produce light. You've got to put energy into this reaction at the reductive recycling stage. So this is a generalized bioluminescence reaction. 

And in fact, all organisms do something that can be summarized or generalized by this. Now, that light that's produced can either be produced in the color that luciferase has been evolved to produce or that light can be used to excite another molecule, what's called a secondary emitter, and that other molecule will then emit its characteristic light. And this is just a process called fluorescence. It's actually happening probably all around us, but in bioluminescent organisms it's a relatively rare thing. Usually most organisms make the color light they want. But if you want to be able to change the color light you make you put a secondary emitter there that takes up the energy of the first light emitter, that is the luciferase, and converts it to another color. Now those of you that know anything about biochemistry in the last 20 years know that there's a molecule called GFP, or green fluorescent protein, that's become very, very important in a lot of molecular and biochemical analyses. That green fluorescent protein was actually discovered by Jim --, an invertebrate zoologist who was studying bioluminescence in the intertidal in California. And it was being produced by a bioluminescent echinoderm, sort of like a starfish but not quite. And that GFP was isolated and is now being used in scientific studies all over the world. 

And it's an example of a secondary emitter taking blue light and converting it to green light. So what about these luciferins? I told you that bioluminescence evolved many, many times in the biological world. And in fact, I think that's really clear from looking at all the different chemical structures that have been evolved to serve as luciferins in different organisms. So you can see that certain species of shrimp and dinoflagellates, remember those algae, that's what the structure of their luciferin looks like. Fireflies, that's their luciferin. Ostracods, which are a kind of crustacean in fish, have this structure for their luciferin. And down here we've got the bacterial luciferin which I'm going to talk about a little bit more in a second. But basically all these molecules have the capability of being oxidized by luciferase and then re-reduced and then oxidized again. And at every cycle producing light. Excuse me. 

So I've told you there's this generalized bioluminescent reaction but bacteria also do this reaction but they have a little twist to it. And in fact they have two luciferins, they have two molecules that they're going to oxidize, and one atom of oxygen from a molecule of oxygen, that is from O2, is used to oxidize each of these two luciferins. One is a flavin and the other is an aldehyde. They're both oxidized to their corresponding oxidized form and then have to be reductively recycled. But it's the same process of producing bioluminescence through the oxidation of a luciferase. In the case of bacterial bioluminescence, it turns out the secondary emitter actually, that some species have, produces a yellow light due to the presence of a yellow fluorescent protein. And that was discovered a number of years back in which here you can see streaks of bioluminescent bacteria. The normal one or the color that you see 99.999 times out in the biological world is that blue light over there. But there are some strains of luminous bacteria that have this YFP, this yellow fluorescent protein, and they'll produce a yellow light as opposed to the blue light. Once again, we don't know why these particular bacterial strains want to produce a yellow light rather than a blue one. 

 So next I'd like to tell you a little bit about the link between symbiosis and bioluminescence. I want to start bringing these two concepts together a little bit. And to do that we need to think about, in particular, why bacteria make light and how that light can be used by the animals that recruit those bacteria in symbioses. And we really don't have a very good idea of why bacteria make light except from the standpoint of their being involved in beneficial symbioses in which the animal gets the benefit of the light and the bacteria get the benefit of a place to live and nutrients that are being produced by the animal for throughout the entire lifetime of the animal. But there have been some other possible functions that have been proposed, and there's beginning to be some information to suggest that actually the bioluminescence reactions can be used in certain pathogenesis, it's been used in developmental signaling, and Margaret may have spoken about that last week, it also can be used to maintain very low oxygen concentrations in tissues because, in fact, the bioluminescent reaction uses oxygen. And also there's some evidence that by producing light, bacterial cells can reduce the extent to which they're sensitive to UV DNA damage. Actually it allows them to repair UV damage more quickly if they're producing bioluminescence. I don't have time to talk about all of those, but I do want to talk a bit about symbiosis and, in particular, how we are beginning to learn that light is being used by animals and how those animals find the luminous bacteria that they need to find. So about 30 years ago we discovered that in fact although there's probably a dozen species of luminous bacteria present in the marine environment, and as I said hundreds of species of fishes and invertebrates that utilize bioluminescence, it turns out that there is a specificity between the species of a bacteria that are found in a given species of host. And, in fact, there are a number of different species that I've got listed up here, I've got four of them. 

Vibrio fischeri, Vibrio logei, another genus called photobacterium leiognathi and photobacterium phosphoreum. And we know that each of these species is found associated with a given family of fish or squid and I've listed over there. There are also some unculturable and undescribed species of bacteria that are found in certain other animal species. And remember the bacteria found in the lure of the anglerfish, anglerfish are called ceratioids, and those bacteria cannot be cultured, at least at this point. We don't know why. If we knew, then we could culture them. (laughs) Sorry, that's not true. But we'd love to know why. In fact, there's no reason to believe that they can't be really, they're not genetically reduced, they have a complex genome. The anomalopid fishes, those are the flashlight fish that I was telling you about that had luminescent organs under their eyes. 

So far a lot of people have tried to get those guys to grow and nobody has had success with it. But you can grow all of these other species up here very easily. They're great bacteria to grow in the laboratory. The only question is, can you maintain the animal as well? And so you can have both the animal and the bacterium growing separately in the laboratory. And some of those species of animals you can, but many marine animals, especially marine fish, are very difficult to maintain throughout their life cycle in the laboratory. They're not like goldfish. You can't get them to easily spawn and lay eggs and go through another generation. Every tropical marine fish that you go and buy at an aquarium has been individually caught as an adult or as a small fish and sent to be sold. As soon as he dies, that's the end of that line. Somebody has got to go out and catch another one and sell it. 

Nobody raises marine tropical fishes, at least not most of the species. So what I'd like to talk to you about for the rest of time is this species Vibrio fischeri and the sepiolid squid and monocentrid fishes that they're associated with. So we're going to be talking about those two hosts and this bacterium, this bioluminescent bacterium. So let me tell you a little bit about this squid. And this is the squid that Margaret talked to you about, for those of you that were here yesterday, last week, excuse me. You can't see him very well, but he's about this big, at least as an adult. And somebody is holding him in a hand up there in that picture. And if you turn him over on his back, you can see this bioluminescence, this little blue spot of bioluminescence down there underneath on the ventrum of the animal. But as I mentioned the monocentrid fishes, they are called pinecone fish for obvious reasons, the monocentrid fishes also have bioluminescent organs. Those organs are right down here underneath the jaw. 

And they actually go along in the reefs at night and you can see the light coming out of these down on the sand underneath. And they're sort of like landing lights on a helicopter, and they're just going around, cruising around, looking down and seeing if they can find worms that are coming up out of the sand and they grab those worms. So that's what they're clearly using their light for as well. They all have this bacterium Vibrio fischeri here. Both of these species, very different animals of course, have Vibrio fischeri as the source of light that's being used to produce the behaviors that they want to be able to produce. So let me tell you a little bit about where you find the sepiolid squids. They're found pretty much all over the world. There's a whole series of species that are found in the Mediterranean, and in fact they're a food fish in the Mediterranean and are used as, well, they actually cook them up in a pink sauce, it's very good. These are a relatively small animal, about an inch long. There's also a species that's found up in Japan. 

One is found down off of Australia. But the species that we work with are ones found in Hawaii. And this is Euprymna scolopes. It's a small benthic squid. It's found in just a few feet of water in the tropical reefs around Hawaiian islands. And down here in the lower right you can see these reefs. These are white sand covered reefs that are surrounding this small island called Coconut Island in Kaneohe Bay in Oahu. And so all around in those sand flats at night you can find this sepiolid squid, this Euprymna. So if you go out there and you gather them up at night, I was just out there last week and after four days of three of us collecting for six hours at night we came back with 21 animals. So you've got to be very patient out there. 

It's not all fun. We were able to bring those animals back to Madison, Wisconsin, where they, I'm sure, feel very disenfranchised. And they're kept now in a sea water aquarium right over in the microbial sciences building across the street and down the road. And what we can see over on the, these are the aquaria where we keep them. They're just recirculating artificial sea water. The animals lay eggs. And in each one of these cylinders are a set of eggs, about 200 to 300 eggs that a single female has laid. Those eggs, you can see here up close. And that's just what is called a clutch of eggs, in this case attached to a piece of coral rubble. And after about three weeks those eggs will hatch into juvenile animals about a millimeter or two in length. 

And those animals hatch without any bacteria associated with them. So they're just like us, they come into the world completely friendless. And they've got to go and find bacteria that will help them live their lives for the rest of their lives. So how do they do that? Well, it begins when the squid first hatches out of the egg and starts ventilating across its gills. Now you remember the way squids work is this: they pull water into their mantle cavity and then they shoot it out through the siphon and that's how they squirt themselves along, that's how they move along. And if we look at an animal, flip him over on his back and draw a cartoon of him like we've got here. You can see the water comes in through this mantle cavity. Here's his two eyes up here, fins down there, that's his rear end. Water comes into this mantle cavity and then goes across this structure in here. It's just a confocal microscopic image of the nascent light organ, the organ that's going to become the light emitting organ. 

And the water passes over, there you go, the water passes over that organ and then shoots out through that siphon. And there's also gills down here, and that's what they're really pulling the water in is to ventilate across their gills and get oxygen into their blood system. But as that water is moving over, it's passing over the arms on this nascent light organ. These are just ciliated epithelial structures that are laying on the top of this nascent light organ. And the Vibrio fischeri is present in the sea water all around these animals. And somehow or other those animals are able to pick that one Vibrio fischeri species out of the hundreds of species of bacteria that are normally present in sea water. And that species get it to colonize its light organ. And we didn't know much about how that was done but Margaret McFall-Ngai's lab discovered that it actually was a very interesting and complex process that happened in the first few hours after the animal hatches. Basically what happens, if we look at this picture down here we'll see this is the nascent light organ, it's just been stained red in this case, and this is a pore on the surface of that organ. And this pore leads to epithelial lined crypts deep inside the light organ. 

And the bacteria somehow or another have to get from the water outside into those pores and into the deep crypts where they're going to grow for the rest of the life of the animal. And so we gave Margaret's lab some labeled bacteria, they were actually labeled with some green fluorescent protein, like I told you it's a very handy way to label biological material. These bacteria are producing their own bioluminescence, but they're also producing their own green fluorescent protein which allows us to easily observe them under the microscope, under a fluorescent microscope. And so what we see here is a whole aggregate of several hundred Vibrio fischeri cells that have aggregated right above that pore. And we didn't know why they were aggregating there until we used another stain that allowed us to see that, in fact, there was a big wad of mucus that had been produced, come out of that pore and sat right there. And it was that mucus that these bacteria were sticking on. And that's how the bacteria that went swooping through the mantle cavity were being held on to there above the nascent light organ. Now, what was really cool was those bacteria, they sat there in that aggregate for a couple of hours, but then they started to dance their way down from that aggregate into the pores. There's a pore here, there's actually three pores on either side of this light organ. And they go down into those pores, into the pores, go deep into the tissues. 

Here we're now looking into the tissues of this light organ, and then a few cells that get in there begin to grow and produce a large population of Vibrio fischeri cells that then produce light. And so this is the process, and as you can see it takes only 12 hours for this whole process to occur. So it's not only specific, it's very fast and a very effective way to make sure as soon as possible those animals have a light emitting structure. So I've been saying it's specific, but what's the evidence that this is a specific association? Well, if we take a section, a cross-section through this nascent light organ, what we see is here's that pore that they're going to go into and we see that the pore is connected by a duct into the deep crypts. Here's another crypt that you can't see the pore that leads into that one. If we look at the animal as soon as it hatches, this is what it looks like, big empty crypts waiting to be colonized. And if Vibrio fischeri are present in the water, within 12 hours a few cells will have gotten in here and all of these little dots that I hope you can see down here, all of these little dots are bacterial cells that have grown up inside that light organ and are now producing light. If we put them in water that doesn't have Vibrio fischeri, it has all the other bacteria that are normally found in sea water, and look at them after 12 hours, after 12 days, after 12 weeks, they continue to look like this. In other words, no other bacterium is allowed to come in there and allowed to grow in that tissue except this one species, Vibrio fischeri. So these animals are extremely good at telling the good guys from the guys that don't belong there. 

We're doing this ourselves, we just don't know how we're doing it. And we're hoping by studying this squid we'll get a clue as to what some of the mechanisms are that animals can use to understand and determine which bacteria they should be allowing to live in their tissues. So this is just to convince you that we looked at a lot of species, all of these are luminous bacteria. They're all very closely related. But, as I say, only Vibrio fischeri can colonize the squid. This Vibrio logei is very closely related. Actually, it's a new species that used to be called Vibrio fischeri but it's a subgroup. And some of the species can colonize but not very well. So clearly Vibrio fischeri is the one organism that can grow well in this animal. And so the reason why we only find Vibrio fischeri when we go out to the natural world in these sepiolid squid is because that's really the only thing that can grow there. 

It's not just that Vibrio fischeri is the only thing that they see out in the natural world. It doesn't matter what they see, Vibrio fischeri is the only one they're going to be able to set up this association with. So how they set up this association is what I'd like to finish up talking to you about. And this is the work of a number of people in my lab, in particular a former postdoc named Mark Mandel who's now assistant professor, starting in January, down at Northwestern University in Chicago. But there's a whole series of people in the lab who have contributed to this work. So in order to talk about this I need to tell you a little bit of, I need to use a little jargon. And it's going to be the names of the bacteria that I'm going to use, the strains. Because it's too hard for me to say squid symbiont strain and fish symbiont strain. So what I'm going to do is, the squid symbiont strain we're going to talk about is ES114. ES stands for Euprymna scolopes strain number 114. 

So that was a strain that was isolated from a Hawaiian squid in 1988. But, as I mentioned, there's a fish species, a Japanese pinecone fish called Monocentris japonica that also has Vibrio fischeri found in its light emitting organ. Separately evolved, completely independent and completely different light organ, not to mention very different kind of animal. We isolated a strain of Vibrio fischeri that we've called MJ11. So MJ is Monocentris japonica. So when I talk about MJ11, I'll be talking about the fish symbiont, and when I talk about ES114, it will be the squid symbiont. So I apologize for that but it's a lot easier for me to talk about. So what Mark did was a very interesting experiment. He wanted to know whether or not the specificity that symbiosis had, that these bioluminescent symbioses have extends not only to a given species of Vibrio fischeri but, in fact, certain strains of Vibrio fischeri. And the question, basically, he asked was, can a symbiont from a fish be able to colonize a squid? In other words, we know that a squid symbiont can colonize a squid but can a fish symbiont colonize a squid? Well, how do we know a squid symbiont can colonize a squid? Very simple experiment. 

You throw the newly hatched squid into water that has whatever strain of Vibrio fischeri you want to put in there and you ask after about 24 hours whether or not that animal is making light. If he's making light, then he's been colonized by the bacteria. If he's not making light, then he's not been colonized. And you can also count the number of bacteria present in the light organism by standard microbiological techniques. And so what Mark did was a very simple experiment where he first showed, as we knew, that if he put the squid symbiont into the water, within about 24 hours there would be somewhere between 50,000 and 100,000 Vibrio fischeri cells in that light organ of that squid. Now this is just to convince you that if we don't put any Vibrio fischeri in the water, there are no bacteria in that light organ. However, if he takes the fish symbiont, MJ11, at the same concentration and puts it into the water and leaves it for the same amount of time, none of the animals become colonized by this other strain of Vibrio fischeri. Even if he uses 10 times as many of this strain, he's not able to get any of the animals colonized. I didn't mention it, but each one of these circles is an individual squid. And we're asking how many bacteria are present in the light organ of that squid after 24 hours. 

So this was the first evidence that there's a real specificity even at the level of strain even within the species. And that was very exciting to us because these two bacteria were closely enough related that we might be able to find what was the difference between the fish symbiont and the squid symbiont. What was the thing that was allowing the squid symbiont to colonize. And so Mark realized that he needed to look and see what were the differences between these two strains, and he did it from a genomic point of view. We had the genome sequence of these two strains, and with the help of Nicole Perna here at the university he basically aligned those two DNA sequences right next to each other and asked what were the differences between those two sets of sequences. Turns out Vibrio fischeri has two chromosomes, one and two, but what we've got here is a depiction that allows us to ask what are the differences as we go along the DNA sequence between the squid symbiont here, chromosome one and two, and the fish symbiont here, one and two. And what you can see very easily is that there are gaps in one sequence, like in the fish symbiont sequence here, that have a gene in them here in the squid symbiont. And so basically, Mark was able to say, well maybe those are the genes that are important. And luckily there weren't a lot of differences here. And basically only about 11% of the genes, that's actually a lot of genes, but still, 11% of the genes were found only in the squid symbiont and weren't found in the fish symbiont. 

Those genes that were found in both were almost identical in DNA sequence, 98.8%. So there was very little difference in these other genes, but clearly the squid symbiont had another 11% of its total genes that were unique to that squid symbiont. And so he began to look through those genes to ask which ones might be important to the symbiosis. And here's where he got lucky, we all to have get lucky. And it turns out that another colleague of ours, Karen Visick who works down at Loyola University in Chicago, had already discovered that there was a gene that was found in the squid symbiont called RSCS. And it was a regulatory gene. It was a gene that controlled the activity of other genes. And when Mark looked between the genome sequence of the squid symbiont and the fish symbiont, as you can see here, the RSCS gene is not present in the position that it is in the squid symbiont. There's no RSCS gene there. The other genes on either side are identical, but there's a missing gene, if you want to think of it that way, in the fish symbiont. 

And Mark thought, well this might be a good place to look for this specificity factor, the specificity gene. Because Karen had shown that this RSCS gene was, as I mentioned, a regulator, but it was a regulator that was found in the squid symbiont but not the fish symbiont. And interestingly, this regulator activated the gene activity of an 18-gene cluster whose job it was to make an extracellular polysaccharide. Now what's that? That's a capsule, that's a sugar capsule that's on the outside of bacterial cells. And interestingly enough, this SIP cluster as it's called, this cluster of genes that makes this extracellular polysaccharide, that cluster is found in both ES114 and MJ11, it's found in both of the Vibrio fischeri strains. But the RSCS is only found in one, in the squid symbiont. And so he reasoned that the control of that extracellular polysaccharide, those genes, might be the important thing involved in the specificity at the level of strain. So let me sell you a little bit more about RSCS. What Karen and her students had found was, as I mentioned, the RSCS gene actually controlled this SIP cluster which made this extracellular polysaccharide and  basically the presence of that extracellular polysaccharide on the outside of a bacterial cells allowed it to form that aggregate. Remember that aggregate that hangs outside of the pore, that allowed those cells to form and stick to that mucus. 

In fact, here you see those pores, now we're looking down on the light organ, you can see the three pores really well, I hope you can. And here's a little green dot here, that's an aggregate of GFP labeled Vibrio fischeri cells. And that's what the wild type or the normal natural strain of GFP bacteria looked like when it's colonizing the squid. So what she asked was if I genetically knock out, I forgot to mention, what is RSCS, what is this regulator responding to? We don't know exactly what it is but it's some signal that's coming from the host. Something that's telling the bacteria to turn on the production of this extracellular polysaccharide. Karen is working very hard to figure out what this signal is. But what we do know is once that RSCS is turned on we make the EPS. If you knock out that RSCS gene and look at a mutant that no longer produces that RSCS, those bacteria are unable to turn on the SIP cluster, unable to form an aggregate and unable to initiate symbiosis. So this is really a central important gene in colonization of the light organ. So Mark's hypothesis was that the carriage of RSCS may play a role in conferring this strain level specificity on Vibrio fischeri cells allowing a squid symbiont to colonize with squid but any other kinds of Vibrio fischeri strains will not be able to colony to the squid. 

So it allows the squid to be even more careful about what kind of bacterium it has. But he reasoned first if this fish symbiont acquired this RSCS gene from the squid symbiont, if he could take that gene out of the squid symbiont and put it into the fish symbiont, would it be able to control those EPS genes, those extracellular polysaccharide genes? Would it be able to do in the fish symbiont what that gene did in the squid symbiont? If we look at colonies of the squid symbiont, the regular squid symbiont, what we see is that they are all wrinkled. I hope you can see those little wrinkles across the surface. And that's because they're making this exopolysaccharide. It makes them real gooey. However, if you look at the fish symbiont, you can see that they're very glisteny, and they don't make this extra amount of capsule or of EPS that causes them to form those wrinkles. So he asked, what if I put that RSCS regulator into this fish symbiont, will it now look like the squid symbiont? And, in fact, I hope you can see, that it does get all wrinkly. So this is the fish symbiont with the RSCS gene put into its genome. So that told us that, in fact, the RSCS could function in another Vibrio fischeri strain, even a strain that didn't already have that gene. So now he asked a really important that I'm sure many of you have already thought about, what happens to colonization when you put RSCS into this squid symbiont, into the fish symbiont, excuse me? And this is where Mark becomes really lucky. 

With the insertion of a single gene into the fish symbiont, it now allows that fish symbiont to colonize the squid. And not only colonize the squid but colonize it as well to the same level as the normal, the natural native squid symbiont. And so this one gene that's able to not actually code for the exopolysaccharide or code for some sort of signal, but in fact all it is is actually a regulator that regulates an already existing set of genes in there that by having this regulator you now can colonize an animal that you couldn't before. In other words, this is the gene that allows the specificity of a symbiosis to change. He also did a phylogenetic survey where he asked if we look at a whole bunch of Vibrio fischeri strains, which strains have it and what strains don't have it? And what he found, what he did was first have a graduate student in my lab, Mike Wollenberg, do a phylogenetic tree of these Vibrio fischeri strains. They're all Vibrio fischeri coming from different places. And then he asked which of those strains have this RSCS gene. And all of the ones that are within that gray square carry that RSCS gene. All the others don't. And so he could reason that right at this point during the evolution of Vibrio fischeri was where the ancestor of all of these strains here picked up that RSCS gene. 

And this was probably the point at which the squid symbiosis evolved, or soon there after. There are no squid symbionts, Hawaiian squid symbionts, above this line, but there are all of these down here are, or most of those are. So in the future, the kinds of questions we want to answer are is this kind of mechanism, that is not changing the kinds of genes that you can deploy but changing when you deploy them in response to what you deploy those genes, that this may be a major way in which bacteria and host can decide how to pick who should be living with whom. And what Mark is going to continue to look at was asking whether or not the addition of this gene not only allows them to initiate this symbiosis, because remember the data I showed you were just what happens after the first 24 hours, can you get in and grow for the first 24 hours, but to be a successful symbiont you got to be there tomorrow and next week, next month, next year. Actually not next year, these guys only live nine months, but you got to be there for nine months. And so that's a long time in a bacterial cell's life. But is this single gene allowing them to persist just like the normal native strain or are there other genes that are necessary to go through this long-term existence? Also, if RSCS allows another strain of Vibrio fischeri to get into this light organ, will it allow another species of Vibrio to get into the light organ? And so if we put RSCS into a different species of Vibrio, will it be able to colonize the squid for the first time? And then of course, we've only looked at one of about 300 genes that are different between the squid and the fish symbiont and already we've found some very interesting examples of how specificity is maintained. And so I'm sure Mark's going to continue his career mining that information of genomic differences in order to look at other genes that may give us clues as to how bacteria and animals get together in a very specific and measured way. So with that, I'd like to thank you all for your attention and hope you have a few questions for me. (applause)