43rd Annual Killian Award Lecture—Sallie Chisholm (2015)

PRESENTER: Good afternoon, and welcome to the 43rd Annual James R Kilian Jr. Faculty Achievement Award. Before we begin, I have a couple requests. First of all, I'd ask that you silence your cell phones and any other electronic devices. And second, you can see that we're already quite full, and we expect others to arrive, so if you're near an aisle and there are seats in the middle, I'd ask that you please move to the middle of your row.

The James R Killian Jr. Faculty Achievement Award was established in the spring of 1971 as a permanent tribute to Dr. James R Killian Jr., president of MIT from 1948 to 1959 and chair of the MIT Corporation from 1959 to 1971. The purpose of the Killian Award is to recognize extraordinary professional achievement by MIT faculty members and to communicate their accomplishments to members of the Institute community.

The title of Killian Lecture is the highest honor that the MIT faculty can bestow on a colleague. It is our privilege today to be addressed by Penny Chisholm, the Lee and Geraldine Martin Professor of Environmental Studies. Having served as an MIT faculty member for more than 30 years, Professor Chisholm has held joint appointments in the Department of Civil and Environmental Engineering and the Department of Biology since 1993.

As noted by her colleagues on the Killian award committee, Professor Chisholm is a groundbreaking scientist, whose research team made a critical discovery about photosynthetic organisms in the ocean. For many years, scientists studied the oceans as a key source of photosynthesis. But prior to Professor Chisholm's breakthrough, they were unaware of the existence of a microscopic marine bacterium called prochlorococcus. This tiny bacterium has been revealed as one of the most abundant photosynthetic organisms on Earth, responsible for producing a sizable fraction of the Earth's oxygen every year.

A cross-disciplinary collaborator and thinker, Professor Chisholm has been associate chair of the MIT faculty, has served as a founding director of the MIT Earth Systems Initiative, has written more than 200 articles on her research, and has coauthored three children's books that explain ecological principles. She has taught introductory biology to our undergraduates for more than 20 years. Today, the MIT faculty is proud to recognize our colleague Penny Chisholm with the James R Kilian Jr. Faculty Achievement Award.

There's a certificate that goes with it. I'd like to read from it. It says, "To Sally Chisholm, in recognition of achievements in marine ecology and environmental science, decades of interdisciplinary leadership and service, and the inspirational vision she has shared with her students and colleagues at MIT." Congratulations.

PENNY CHISHOLM: Thank you. Phew. Pressure's on.

As this day approached, I kept realizing more and more how significant an honor this is and how terrifying it is to address this group of incredible people, students and colleagues.

So first, before I start my lecture, I want to thank people, because I don't want to run out of time and forget to thank them. So first, I want to thank the committee that selected me and all of whoever was behind all that-- it remains a mystery-- all of the collaborators over the years that have contributed to this work.

I'd like to thank my husband Don, who has to compete with prochlorococcus every day for my attention, and the organizers of this event. Also I'd like to think the funding that I've been lucky enough to get. And so many agencies and families need to be recognized. These are the people that funded the chairs that I've enjoyed, that have allowed me to do basic research, get to just follow your instincts, and the foundation research that I've gotten, which has been wonderful, government research. And, of course, MIT and Woods Hole have been the foundation of everything that we've been able to do.

But, most importantly, it's the students and the post-docs and the staff that are behind this work that I'm going to talk to you about today. I'm going to steal a line from Simon Levin, a colleague where I heard him once say, when I say I, I mean we. And when I say we, I mean they.

And they know that that is absolutely true. My students and post-docs have taught me just about everything that I'm going to share with you today. They've come from many different disciplines over the years. And it's just been a great joy for me to watch them work together.

We work in teams, because to attack this problem-- as you'll see, we work from the level of genomics all the way up to the level of the biosphere. And no one person-- let alone me-- can possibly be an expert at all those levels. So it's through their collaborative spirit that we've been able to do this work. And as I've told them many times, it just gives me great joy to see them working together on these problems.

I'd also like to thank everybody in the Parsons Lab, which is this building here on the corner of Vassar and Main Street that most of you probably never notice. Or if you walked by, you'd probably say, wonder what that's doing here. I call it the hot dog stand in the middle of Manhattan.

And we endure floods. In fact, I was just reminded today during the party to celebrate when I got the National Medal of Science, a huge main broke and water poured right into the middle of the party, which I thought was beautiful. Our motto is ideas trump facilities.

So as I was preparing this talk, I started going down memory lane. And I found this picture, which is the Parsons Lab, across from Building 20, a lot of you students don't even remember that era. But some of the oldsters in here with me do.

There's my parking place. I could park right next to the building. It was a very exciting time back then.

Now, I found this picture, 1978. I came here in 1976-- I will get to the science. I promise. I came in 1976. And find me in this picture. There I am.

And if you could see the bubble coming out of my head, it was, "What am I doing here?" And in fact, if you could see a bubble now, it would be saying the same thing.

I am a biologist by training and oceanographer in the Department of Civil-- it wasn't even civil and environmental engineering then. It was civil engineering. But they hired me. And thank you very much for that. And it's been an incredible journey.

So I decided to do sort of a historic thread for this, to follow the path from that time how we ended up making this discovery of prochlorococcus and what it's taught us. And at the end, I'll try to conclude with what it has taught me about how to think about the life sciences.

So first of all, I always start my lectures with this slide, which is my abridged version of life on Earth, which is the reaction, photosynthesis, where sunlight, energy, carbon dioxide, and water is split, and biomass is created, and oxygen is evolved. And this is the foundation of all of life on Earth.

And if you Google it in our course catalog-- photosynthesis-- you won't find it, which is something that I've been working on for years.

And the reverse of that-- so this is done by all plant and phytoplankton. And the reverse of that is done by everything, because you have to break this organic matter apart by oxidizing it with oxygen to get the energy out. And CO2 and water are released. And that's where we get the energy to be alive.

And, of course, microbes are incredibly important for regenerating all the other essential nutrients for life in this.

And the way I think about this, we know the saying, ashes to ashes, dust to dust. It really should be gases to gases, dust to dust.

The Harvard Smithsonian did a study about education. And photosynthesis was one module. And they actually filmed MIT and Harvard students at graduation. And they said, here's a seed. Here's a log. Where does the weight of that log come from?

None of them could answer that. They danced around it-- the water, the soil. And then they said, if I told you it came from the air, one of the students said, I'd have to think about that.

So it's just this fundamental feature of life on Earth, that all of this biomass, all of life comes from the air. And because we forget that and we don't think about that, our perspective of life on Earth is dramatically slanted.

So to overcome that, I written two children's books on photosynthesis. This one on land. And that one in the oceans, available on Amazon.

And they're children. But there are also for people of all ages. They cover the basics.

So half of the photosynthesis on Earth, roughly, is done by the phytoplankton in the oceans. Photosynthesis and then respiration draws CO2 in. Respiration draws it back. And the same on land.

And even though the mass of the phytoplankton is a tiny fraction of the land plants, they do just about as much photosynthesis. And if you're not used to thinking in terms of gigatons, that's the weight of 50 billion Volkswagens is flying from the air into plant biomass every year to feed the biosphere.

And, of course, this is fossil fuel, fossil photosynthate that we are now burning and putting into the atmosphere, which is causing climate change. And I've written a book on that, called "Buried Sunlight," which is very important reading for people of all ages.

So phytoplankton, they're drawing the CO2 out of the atmosphere into this food web. And then most of them are eaten through the food web. And, of course, that goes right back out into the atmosphere.

But a tiny bit of this carbon that they're drawing in actually finds its way to the deep ocean. And once it's there, it's a sequestered for hundreds to thousands of years, depending on how deep.

So as you look at the CO2 concentration in the ocean as a function of depth, you see there's a huge, highly concentrated CO2 in the deep ocean. If the ocean's mixed surface to bottom and equilibrated with the atmosphere, the atmospheric CO2 would be about double, maybe triple, depending on who I talked to over in EAPS, in the atmosphere.

So the phytoplankton are serving to keep this gradient and pumping CO2 down. They're doing their job.

Here's a satellite image of a phytoplankton bloom off, I think this is, Argentina. And if you go in there and you look, some of these-- these are colonies of phytoplankton. You can actually see with the naked eye. If you're snorkeling in the Caribbean, you can see these little puffs that look like sawdust. Those are colonies. And then if you go down another order of magnitude or two, you see tiny microscopic cells.

And this was our image of phytoplankton. We thought they were all about this size until 1979 or so.

So at that time-- so now we're in 1976. And I was working on different species of phytoplankton. These were what I call my muses. This is a diatom and a dinoflagellate. We were studying various aspects of their physiology.

And my first PhD Student, Neil Shifrin, was interested in working on something that had applications. And I thought, well, that's probably a good thing to do since I'm in an engineering department. And so we got funding from Exxon to study the lipid production in different species of phytoplankton.

And I just in preparation for this talk, I said, I wonder if anybody reads that paper. And I went and I looked at the citation index, which we can do now, and saw that recently there's been a big increase in the number of people that are looking at lipid production in phytoplankton. And then I compare it with the price of oil. And it's amazing.

But it is very heartening that this basic research that we did 35 years ago, now people are looking at it and citing it. So that's exciting.

But as part of that study, we were looking at how these cells grow on light dark cycles. Because people typically in those days would grow phytoplankton in continuous light. And I always thought, why don't they explode? They need rest. It's gets dark in the real world. Why do they grow under those conditions? But they do. They don't mind.

But we were interested in studying their cell cycle. And this, I've just added a couple of nuclei here. So there's a cell ready to divide. And here's cells that have divided.

And in order to do that, we had to use an instrument called flow cytometer. And the biologists in the audience are familiar with these. And back in the day, they were used in cell cycle research and cancer research.

And so the way it works is you inject a sample. And it's focused by fluid. And then it goes through a laser, cell by cell. And if you stain the nucleus, the DNA, you can tell which ones have divided, replicated their DNA and which ones haven't.

And here's where I have to start crediting, Rob Olson and also Sheila Frankel. Those of you in the Parsons Lab know Sheila. Back in those days, she was helping us do our research. And using a book written by Howard Shapiro, they actually built us a homemade flow cytometer. We didn't have the money to buy a real big rig.

And so we were doing this work. And we realized that this instrument was perfectly suited for studying phytoplankton, because they have pigments that fluoresce if you excite them with laser. You don't have to use a stain or anything. They just they just naturally fluoresce from the chlorophyll.

And right about that time, John Waterbury from Woods Hole, in 1979, had just discovered these tiny little cyanobacteria in the oceans that were about two microns in diameter that fluoresced orange. And so we said, oh, that's exciting.

And, in fact, that is the biggest discovery, bigger than prochlorococcus. Prochlorococcus is sort of a me too discovery. But for our purposes here today, I will blow up prochlorococcus. This one is under appreciated.

But anyway, so we said, that's exciting. We can use a flow cytometer to study this. And this where Rob comes in.

He was fearless about taking this instrument that's built to be run in a hospital for biomedical research and put it on a ship under these kinds of conditions. And you're focusing this tiny little laser on this thing. And so this is Rob actually on the ship, looking very frustrated-- the young Rob looking frustrated.

But this is a typical lab in a ship. You have plywood, two by fours that you have to like nail things down and. So it was just a miracle that it worked. But it worked beautifully.

So we were studying this synechococcus, which fluoresces orange. So this is a depth profile. This is forward scatter, which gives you some size. And here's the orange fluorescence on this axis. And so this is different depths.

And you can see these populations of synechococcus jumped out. It was easy. We can study them. We could go long transects. We could count their numbers. It was really exciting.

But then we started noticing something strange. And this is where I credit Rob with this discovery-- because he's the one that noticed this. He was the one that was at one with the instrument-- where there were these little red fluorescing cells. There wasn't supposed to be anything that small that contained chlorophyll, which fluoresces red.

And this was electronic noise. And these guys were coming out of the noise. And as we went deeper, there were more of them. And we thought, hmm, that seems a little biological.

They're increasing with depths. There is no reason for the noise to increase with depth. And there is reason for cells to have more chlorophyll as you go deeper because there's less light. So we thought, hmm, that's kind of interesting.

This is the original trace. We said unidentified picoplankton.

Rob was able to get this picture. So here's synechococcus, bigger, orange fluorescing. And here's two little prochlorococci cells, fluorescing red. And the same picture with a light microscope shows you those two cells so you can see why nobody knew they were there before. They're just look like specks of dust. Now we know what they look like. You can find them.

All right, Jesse, my graduate student, told me I had to put this slide in. Maybe because it makes me look human. I don't know. Or to prove that I actually did go to sea back in the day.

So we're off Bermuda. We were pretending to be prochlorococcus. We made blue glasses for ourselves, because that's the color of light that they see. This is what you do in between when you're going from station to station.

And this is Rob. And Erik Zettler was a key person then, and Ginger Armbrust, a graduate student at the time.

So we published this paper. At this point, we still didn't realize how abundant or significant this would be. We just thought that that was really cool, the smallest-- turns out physically the smallest photosynthetic cell.

They're about the size of the wavelength of light that they collect. A physicist pointed that out to me one time when I gave a lecture. And I've always liked it, because I have this image of a prochlorococcus with these waves going through. And I know that's not what happens. But that's okay.

And they contain chlorophyll b, which was very unusual for a prokaryotic cell. And I don't have time to go into that. But for a while, we thought it was a missing link, sort of a living fossil. And it isn't. But that's okay. But that's why we called it a prochlorophyte. And it also contains this unique form of chlorophyll, divinyl chlorophyll, which allows you to go out and measure what fraction of the total pigment, total chlorophyll, is actually in prochlorococcus.

So recently, Adam Martiny and his colleagues-- Adam studied prochlorococcus as a post-doc in the lab some years ago.

They did this exhaustive survey-- so all of these transects in the oceans. And they calculated how many prochlorococcus there were on these transects. And here's the global map of prochlorococcus. And we've estimated there are 3 billion billion billion cells per planet. I like that unit. So this is by far the most abundant photosynthetic cell on the planet.

And in some areas of the ocean, like the central gyres, they can be even more than half of the total photosynthetic chlorophyll. So it's incredibly humbling to realise that we didn't even know they were there. And, of course, all of our models and images of the ocean worked perfectly fine before we knew they were there. And I continue to be humbled by the things that needs major things that we learn that change the way we think about the ocean.

So the record concentration is 700 million cells per liter that's been recorded. And the estimated global synthesis is equal to the weight of 5 billion Volkswagens. That's just prochlorococcus, how much CO2 they bring.

Okay, so what's their world like? Here's my image of their world. So here are the prochlorococcus cell. They divide about once every day or two. But they're eaten just about as fast as they divide by small protozoans that we really know essentially nothing about. And they live, of course, with hundreds to thousands of different species of other types of bacteria-- here non-photosynthetic bacteria and also all the larger phytoplankton, which they compete with for nutrients.

And it's an incredibly dynamic system with turnover times on the order of days and extremely dilute concentrations of nutrients. We think of them, especially for the trace metals, they see them atom by atom. They bump into them. At least that's the calculations are if you think of it all as one big soup. And the distance between cell to cell is many body lengths. So it's very dilute.

And yet, they're stable. Their abundances are stable over decades from ocean to ocean. So this is a time series. This is off Bermuda and off Hawaii. These are two of our favorite study sites. Prochlorococcus likes warm water, which is convenient.

And so these are two time series. This is 1990 to 1994, and, again, 2004 to 2007. And you can see, there is seasonal variability here. But it's very similar, even a decade later.

In Hawaii, it's a little more stable, because you don't have the huge seasonal changes. But year to year, decade to decade, you have roughly the same stable dynamics-- remembering that this is turning over every couple days. And yet, you can count on them to be there in those numbers, more in Hawaii than in Bermuda, because it's warmer.

We don't know that that's the reason. In fact, that's one of the mysteries. But there are always count on more in Hawaii.

So the big breakthrough was in 1990, Brian Palenik, who was a joint program student on one of our cruises, succeeded in getting a culture of prochlorococcus. Up until that time, everything we learned about it, we had to do at sea, doing experiments with live cells in bottles.

We'd poke them. We'd change the light intensity on them. And then we'd measure them with the flow cytometer and see what happens.

But he managed to get some into culture. So we were able to formally describe them and give them a name.

And that's when I realized that this was what I wanted to do for the rest of my career. Because this is a simple cell, very widespread, you could count on going on a cruise to find it. When I was studying diatoms, you'd study them in the lab. But then you'd go out on a cruise, and you couldn't count on having it there. So you couldn't study it in its natural habitat.

And so they're easily measured at sea with flow cytometer, now called cultivatable. And also more importantly, is it's a microbe whose environment is easily measured, because it's a liquid environment. So you could study them in the context in which natural selection operates, unlike bacteria in soils or in our human gut or where you have this very non-homogeneous environment that you can't easily measure the concentration of various things that influence the cells.

So the first thing learned-- so the first culture came from deep water in the Sargasso Sea, and the second culture from one of my former students Daniel Bellott and his student Patrick-- I was going to the Patrick Jaillet. But it wasn't. I don't think it was you. It'll come to me. He's French and his name is Patrick. My students will tell me in a minute.

Any way, he cultured the second one, which was from the surface.

So when we started studying their physiology, Frederick Partinski was his name. So it wasn't Patrick.

We started studying the physiology. The first thing we learned is that they had different optimum light intensities. So this is what we call a high light adapted strain, which would grow optimally at light intensities that would pretty much kill the low light one. And then the low light adapted strains could grow at light intensities where this guy couldn't even make a living. As we isolated more and more, we found that they'd fall into these general categories of high light adapted and low light adapted strains with small variations.

And then we were able to develop techniques where we could measure their distributions in the wild. So here's a depth distribution. Here's a low light adapted strain. And here's the high light adapted strain.

You see this one is by far-- I don't have a scale on here. But it's orders of magnitude more abundant than the low light adapted strain in the surface. But as you get down here, this is where these guys thrive.

So then when we started looking at comparing these two high light adapted strains. And this is a transect Zachary Johnson, a post-doc at the time, did on along the Atlantic Ocean, showing the relative abundance of these two high light-- I just call them the greens and the yellows along this transect, showing that they have very different abundances.

And so we go in the lab, and we look at those strains. And, of course, what we find is that they have different temperature adaptations, as a growth rate, as a function of temperature. So this one likes the equatorial waters, the high temperature, where this one can grow at low temperatures, where this one can't. So we have two environmental variables, light and temperature, upon which these organisms differentiate their niche.

Then as the years went on, we learned to develop techniques that we could measure all these different ecotypes, as we call them, on this time-- this is a time series off Bermuda. Here's the log scale showing this high light adapted one is by far the most abundant. This is the green high light adapted one, and then three low light adapted strains.

And you can see that they oscillate in abundance with the seasonal changes. But they oscillate out of phase. They have specialties.

This one, for example, which Jesse Thompson, one of the graduate students is becoming an expert on, is actually better adapted to fluctuating light, when the water column mixes in the winter all the way down from 100, 200 meters. It's one that seems to thrive. And it can tolerate that kind of light mixing, whereas many of the other ones don't. Like these guys, they can't take it. So their abundance really goes down.

One of the things we notice here is that even though these are orders of magnitude more abundant than some of these low light adapted strains, these are always there. We're tempted to say, oh, these are the most important and blah, blah, blah.

But they're all important, because they're always there. If you're there next year, you're important. You're doing just fine. And they always are, next year.

So with that we had studied them on two-- this is the formal definition of the ecological niche that was put forth by G. Evelyn Hutchinson some years ago when I was in graduate school, that the ecological niche is an n dimensional hyper volume, every point on which an organism can survive and reproduce, which, of course, means it's not measurable.

It's a hypothetical construct. But we try to measure some of those dimensions, the ones that we think would be the most important in controlling the distribution and abundance of the cells.

So we had two dimensions. And now we have many, many more to look at. And it was about this time that genomics entered the scene.

And we were lucky enough to have-- I think prochlorococcus was one of the second microorganisms to be sequenced, because we could sell it because it had a very small genome, and it wouldn't take that long to sequence. Not that long back then was about a year.

Too long now my students think is two hours. It's just amazing how things have changed.

So I managed to convince the Department of Energy that was sequencing these that the bang for the buck if they sequenced two, comparing a high light adapted and a low light adapted strain would be much greater. And this was very greedy of me because everybody at that point wanted the sequence of their genome. But I figured two prochlorococcus is equal to one something else, bacillus, because they're small.

Where's Alan? He's going to shoot me.

But they are very small. And it wouldn't be as hard to sequence two very similar things, etc.

Anyway, it was very exciting. And it took a while. And that was when I was getting way out of my depth.

I remember distinctly, Gabrielle Rocap was a graduate student in the lab. She had come from the MIT biology department. And I remember going in and saying, Gabrielle, if we're getting into this, you're coming with me. And I said, I'm not going to do this without you. Because I didn't know anything about genomics at all. I barely knew much about molecular biology.

So we started the adventure. And with the help of all my students, we got our first genomes.

The first thing we learned was that the genome sizes were dramatically different, the numbers of genes that these two strains. The low light guys had had significantly more genes. And that there was a smaller suite of genes that they had in common, 1,350 that they both had. And then the rest were different. And they also had different base pair composition for those of you who care about that kind of thing.

And if you lined up the genomes of these two strains, if they were identical, there'd be a perfect line here. They'd all have the same genes. And they'd be in the same place.

Well, you can see that they aren't even close. And then all these ones on the axes are genes that one has and the other doesn't have. And then there's rearrangements. These are very different. And by any standard definition of a species in bacteria, this is a single species, these two guys.

So that was sort of in my mind the birth of ecological genomics for us, where we're able to map the gene content of cells onto the environment from which they've been collected. So the cells from the high light adapted cell had many more light protective proteins. These are just examples. The low light adapted cell had enhanced pigment synthesis.

They had different sets of nutrient acquisition genes. Because there are these gradients in the oceans, low nutrients in the surface, high in nutrients in the deep water. A temperature gradient here sets up as a shallow mix layer where you have regenerated nutrients by this turnover time of organisms being eaten. So that was exciting.

And this is Gabrielle Rocap was the student who I just spoke of that this was the first author on that paper.

And it was about that time that it suddenly dawned on me that prochlorococcus was really special. I mean, we already knew it was special. But now I think of it as it's absolutely the minimal life form. It's the smallest amount of information-- 2,000 genes on average worth of information-- that can convert solar energy and just inorganic compounds. There's carbon dioxide, air; inorganic compounds, rocks, if you want to think of them that way.

They don't require any amino acids or any kind of organic carbon that is manufactured by some other organism. So they are really the essence of life, an incredibly simple, elegant little cell.

So we have a bit of a handle on the diverse-- we're starting to get a handle on the diversity of this bug. And so we start to wonder how diverse are they globally?

So is my mental image of-- when I look at the oceans now, I just see the millions of prochlorococcus cells. And for years I'd say, I want to within a milliliter of seawater how different are all those prochlorococcus cells in that milliliter of seawater?

So recently, now that you can just sequence a genome in a day, I know, the students-- easy for me to say, just sequence a genome. Steve Miller, post-doc in the lab, when he came, I said, we have all these cultures, just sequence the genomes. And it's a lot more complicated than we thought. But it was worth it.

And so now there are 40 genomes. Every time we sequence a new genome, it quickly settles down to these core genes, which they're about 1,200 genes that all the cells have. Every single cell has those same genes. That's the essence of being a prochlorococcus.

But every cell has roughly 100 to 200 new and different genes. So the number of genomes as you do pairwise comparisons, you keep accumulating more unique genes in this prochlorococcus gene pool, so-called the pan genome.

And if you do a projection just from these cultures, theoretical projection-- which is fraught with difficulty, but you've got to start somewhere-- is that the total gene pool of prochlorococcus is 80,000 genes in the oceans. We humans have 20,000 genes. So this is like a super organism. Collectively, it has an incredible amount of information.

So here's a picture, which you'll see more of it as I go forward, where this little squiggle represents the core genes. And then these stars represent these flexible genes, that differ strains or whatever you want to call them have different flexible genes.

And when I was putting this lecture together, we had a group meeting-- I don't know if Katya is here? Anyway-- yeah, she's way up there.

Okay, so I always consult with my people. And I said, I need an analogy. Because when I give us talk-- I've given this talk to other people-- and they say, yeah, like blue eyes and brown eyes. And I said, no, the blue eyes, brown eyes, it's the same basic gene. We all have the same number of genes, more or less, we hope.

But these are totally different genes. They do totally different things. So I said, I need an analogy. And Katya came up with iPhone. And it works really well. And you can spend an hour talking about this analogy.

So the species as an iPhone-- and you could have a different one, whatever somebody else uses. I don't know what the other one-- Samsung or whatever. And when you buy them, they come with these core apps. You can't even get rid of them if you want to. Did you ever try to delete some of those? I don't want half of these, but I can't get rid of them.

But then there are all these other apps that influence your fitness depending on your environment. For example, if you are very thin and never gain weight, you don't need this one. The person that has the flashlight app, when it's dark and has lost their keys are going to have an increased fitness over someone else. These games could increase or decrease your fitness, depending on how much time you spend on them.

But the combinations of these apps would define a strain. And I looked this up. There are 1.2 million flexible apps in the global app pool. So the ratio of flexible to core for the iPhone is much higher than in prochlorococcus, but the same general idea.

So these flexible genes that the different strains have, we think of them as reporters. They're telling us what the selection pressures are on that strain in the environment from which we got it. So they're telling us something about the oceans and how they work.

A lot of them are involved in cell surface properties that would implicate predators or viruses that infect them, which I'll tell you about in a minute. Many are involved in the acquisition of specific nutrients that vary from place to place in the oceans. And many are involved in various stress responses.

It's very interesting that many of these, most of these, flexible genes have been acquired from very distant phyla. They're not from cyanobacteria, which is the broad group that prochlorococcus belongs to. So there's a lot of gene exchange going on in this pool in the oceans.

So we next started to wonder, well, how are these flexible genes distributed throughout the oceans? What are they trying to tell us, the prochlorococcus as they hold on to them?

And about this time a tool of metagenomics was being introduced to our field. And actually Craig Venter had taken his yacht and sailed across the oceans, the warm water oceans, where prochlorococcus loves to live, and sampled all this DNA, so with a tremendous amount of data for gene sequences, just pieces of sequences of bacteria from all over the oceans.

And with genomics-- so these would be the chunks of bacterial DNA, that would be everything that's there. But if you have cultures, which we had a lot of then, and their genomes, you can by homology drop-- you can pull out the genes from this sea of genes that belong to prochlorococcus. And then using that set of genes, you can say, well, what are they like in this environment? What are they like in that environment?

And so the first thing we did was compare our favorite sites, Hawaii and Bermuda. We could ask the simple straightforward question, of all the prochlorococcus in Bermuda and Hawaii, how did their gene sets differ?

And it's a much longer story than this, but this is the work of Maureen Coleman, a graduate student then, was able to show that the prochlorococcus in Bermuda had a much higher frequency of phosphorus acquisition genes than the ones in Hawaii. In fact, that was absolutely the strongest signal that came out of the comparison of the gene pools.

And it makes sense, because the phosphate concentrations here are order of magnitude lower than here. So these prochlorococcus over evolutionary time have evolved various mechanisms to scavenge more forms of phosphorous from their environment, because that's a really strong selection pressure. And again, the cells have much to tell us about how the oceans work.

So we're getting more and more diversity we're finding. So just getting to that question of how much diversity is there in this milliliter of seawater if we collect it? This is when we started working on single cell genomics. And it's the flow cytometer again that made this possible.

Here is the flow cytometer. And I didn't show you this dimension of it before. But as the cells go through this capillary tube, and then there's a vibrating piece here that breaks the fluid stream into droplets, and you can set it so that each droplet contains a single cell. And it still amazes me that this works. It works amazingly well. And it just blows my mind.

So they go past the laser. The laser talks to a computer. The computer says, I want that one. And then there are these charged plates. And it would charge the droplet, which will deflect it out of the stream. And you can put one in each little bucket here.

And then you can amplify its DNA. And then you can select the cells that you want to sequence. And then you sequence genomes of individual, tiny little prochlorococcus cells.

Now, I'm making it sound really easy. And it's not. It took us years. And we did this work, ultimately, we did in collaboration with a colleague at the Bigelow Labs, where they have a facility that does single cell genomics.

So this is the work of post-doc Nadav Kashtan looking at a single prochlorococcus cells as they change over the season in Bermuda, our Bermuda site again. And to cut to the chase, and this was a really enormous amount of work, both in the lab and in terms of the bioinformatics, there is extraordinary cell to cell diversity as we look just within these samples.

Back to our iPhone analogy, to understand that diversity, what Nadav was able to show is that there's even diversity in these core genes, in the core app. Now the analogy breaks down a little bit. Maybe if I understood more electrical engineering, I could talk about how your app, map app might be slightly different from my map app, maybe a little faster or I don't know what. But they're slightly different but fundamentally the same function. And then they're coupled to flexible genes.

So Nadav identified these backbones populations that were a set of core genes with particular features that were coupled to particular apps. And there were thousands of these backbone sub-populations. Each of them, we estimate with trillions of cells belonging to them.

So the picture that now has emerged, if this is the seasonal cycle of total prochlorococcus off Bermuda, and then I showed you that within that total population you had these light and temperature ecotypes oscillating in different abundances. And what Nadav was able to show is that within those, there are hundreds of different variants.

And it's not just random. You can through various analyses show that these are the product of selection.

So recently we published a paper in Nature Reviews. This is a paper by Steve Biller. And this was the cover they put. We call them a prochlorococcus federation.

So this is the federation of many different strains. Why they have them marching on land is beyond us. But you get the idea. I love that they each have their own flag. And they go on forever.

So enough of that diversity. Now, I'm going to shift gears a little and move into a cautionary tale. And given all the diversity that I've talked about, I want to tell a story about what happens when you isolate a particular prochlorococcus and start to build stories around what that cell is like.

But first, we want to understand a little bit about nitrogen in the oceans. Nitrogen is thought to be a key limiting factor of ocean productivity. If you take seawater from one of these open ocean gyres and add a little phosphorus, you might get a little phytoplankton growth. If add phosphorous and nitrogen, you can create a bloom. So nitrogen is considered one of the primary limiting factors in the oceans.

Here's some bottles being incubated on a ship.

And there's much more nitrogen available than the ammonia in the ocean. So it's a primary source of nitrogen.

And it turned out that the first prochlorococcus cultures that we isolated, none of them would grow on nitrate. And that was just really, really puzzling.

And in fact, I remember when we started talking about this, Jim McCarthy, who's a colleague at Harvard, who built his career studying nitrogen metabolism in marine phytoplankton, called me up and he said, what is this prochlorococcus doesn't use nitrate? Because all phytoplankton are supposed to use nitrate.

And we learned when we got the genomes that they couldn't use nitrate because they're missing an essential gene that's necessary to reduce nitrate to nitrite then to ammonia so they could use it in biosynthesis. And this was particularly puzzling because one of its close cousins, synechococcus, the one that I'd told you earlier was neglected, could grow on nitrate, and was known to grow on nitrate.

So when you discover something about like this about prochlorococcus, because the multiplier is huge-- there are 10 to the 27th prochlorococcus cells per planet-- that has a huge effect on the nitrogen metabolism of the oceans. So the causal implications-- so nitrate affects phytoplankton growth. That affects CO2 flux from the atmosphere to the ocean. That affects CO2 in the atmosphere. That affects global temperature.

So this picking this one cell had huge consequences in terms of how we interpret how the biosphere works. Not that people were worried about it. But I was.

So I don't have time to tell you the whole story. But we had reasons to believe there were prochlorococcus out there that could use nitrate. John Casey, a fellow working off Bermuda, had shown if you add nitrate to seawater with a label on it that, and then you sort out the prochlorococcus, it was in the prochlorococcus.

And so Paul Berube in the lab started looking at the metagenomes in the field and was able to find this gene that was missing from our cultures attached to pieces of prochlorococcus DNA. So he was able to say, they've got to be out there. And then went on the hunt with using media with nitrate in it to try to select for them and was able to.

And so this is an ongoing mystery. So far some of them do. And some of them don't. And we don't know why. And we're trying to understand what the selective pressures are to make some of those cells hold onto that gene and others let go. And one of the ways we do this is study their distribution along gradients in the ocean.

So prochlorococcus is not alone. One of the other things that we do in studying it is try to study how it interacts with the rest of the organisms in its environment. Here, I'm going to just tell you one little story about interactions with bacteria.

For years, we had real trouble getting prochlorococcus free of the bacteria that like to grow with it. Here's prochlorococcus cell. And here are heterotrophic bacteria that require carbon that like to grow with it.

So a clever graduate student of Erik Zinser figured out that one of the reasons that prochlorococcus is feeding this bacteria, organic carbon, and the bacteria is providing the function of reducing the oxidative stress of prochlorococcus. Prochlorococcus is missing this enzyme, which is necessary to reduce the oxidative stress. And this cell supplies that function to it.

And this is just one interaction. But what's interesting from this is that they have developed a whole theory of evolutionary dependencies using evolutionary game theory just from studying these interaction. So I offer it as an example of how just poking around in the lab and studying what would seem to be trivial little things can lead to major changes in the way we think about the evolution of life and interdependencies of organisms.

Here's another thing that prochlorococcus showed us. This is Steve Biller, postdoc in the lab's work. We noticed for years these little bubbles on the prochlorococcus cells. Every time somebody came to the lab, I'd say, what do you think these bubbles are? You want to work on that?

Most people said, I don't know. I don't care. I got some I know what I want to do.