Session 2: Microbial Diversity and Evolution

00:00:02.17 Hello, and welcome to iBioSeminars. My name is Dianne Newman, and I'm a professor in the Divisions of Biology
00:00:07.16 and Geology and Planetary Sciences at the California Institute of Technology,
00:00:11.26 and I am also an investigator at the Howard Hughes Medical Institute.
00:00:15.13 So I am going to be giving you a lecture in three parts today,
00:00:19.16 and this is part one, which will be a very general overview on microbial diversity and evolution.
00:00:25.09 In part two, I'll tell a specific story about a modern example of a microbial metabolism
00:00:30.29 that's quite interesting and very important in affecting the geochemistry of the environment with
00:00:36.04 regard to arsenic geochemistry.
00:00:38.06 And in part three, I'll talk about work we've been doing that is more directed
00:00:43.17 at understanding a metabolism that evolved in the past, namely oxygenic photosynthesis.
00:00:49.02 But let's start with an overview now
00:00:52.14 and consider four important points about
00:00:57.00 microorganisms and their history. And I am going to walk you through each of these four points.
00:01:02.14 So the microbial world is really quite remarkable
00:01:06.23 and my goal in this first overview is to leave you with an impression
00:01:10.22 of its diversity, its antiquity, and how abundant and ubiquitous
00:01:16.18 this world is. So let's begin with antiquity.
00:01:20.27 When we think about the evolution of life, oftentimes we think in terms of macroscopic
00:01:25.23 fossils such as the ones that you see here. And it is pretty clear when you look
00:01:30.01 at these rocks, that something living was present on Earth when they formed.
00:01:33.24 In this panel over here you see a fossil of some type of algae. It is not clear
00:01:39.26 exactly what type, but it is inferred to be an alga.
00:01:42.09 And this shape here is known as a trilobite.
00:01:45.06 And this section of rock that you are looking at is one of the most famous fossils on the plane.
00:01:50.12 It is called the Burgess Shale. It's found in Canada, and it dates to what we call the Cambrian explosion,
00:01:57.14 which occurred roughly half a billion years ago around
00:02:00.29 five hundred and sixty million years ago.
00:02:04.16 So we can certainly claim when we look at rocks of this age that life was present.
00:02:09.17 But if we want to think about the evolutionary history of life,
00:02:12.22 over a much larger time span of billions of years,
00:02:15.26 given that the Earth is 4.6 billion years old,
00:02:20.02 we need to step back in time and look at more ancient rocks.
00:02:22.12 And when we do this, the shapes suddenly change, and it becomes
00:02:26.03 not quite as evident that we are looking actually at fossilized versions of life,
00:02:30.09 and yet we are. So for instance, take this rock as an example.
00:02:34.07 Here you see these dome-like structures, and these are vestiges of a type of microbial community
00:02:41.29 forming in a shallow marine environment
00:02:44.16 that became lithified and left these domal structures,
00:02:48.07 and we call these structures stromatolites.
00:02:50.08 Now this particular rock that you are looking at
00:02:52.21 is about 3 billion years old and is from South Africa.
00:02:56.19 But these rocks can be found all over the world, and they occur
00:02:59.21 throughout Earth's history, going back as far as 3.4 billion years.
00:03:04.20 However, when we go even further back in time,
00:03:08.04 for example, back to 3.8 billion years,
00:03:10.17 you can see ore deposits that one might not intuit immediately had anything to do with
00:03:16.26 microorganisms, and yet they do. They indeed record a history of microbial activities that was quite profound,
00:03:24.24 so profound that it quite literally transformed the planet.
00:03:27.19 And this is one beautiful example. So what you are looking at here is actually a 2.4 billion year old quarry.
00:03:35.27 This is in Western Australia in the Hamersley formation, and this is known as a banded iron formation.
00:03:41.13 And they're extremely important today because they constitute the world's largest source of iron ore.
00:03:47.03 But they also record a remarkable history of the evolution of metabolism.
00:03:51.13 Now how can this be? How do these massive rock quarries tell us anything about microbial life?
00:03:57.14 Well, when you think about what they actually constitute
00:04:01.00 they are made up of iron minerals, as well as other minerals
00:04:04.21 cherts, which is a type of silicon oxide,
00:04:07.07 intermixed with these iron species, but for now let's just focus on the iron.
00:04:12.02 So how did this iron get into this big deposit that you see here?
00:04:15.12 Well, it began a long time ago in ancient seas, in the form of ferrous iron
00:04:20.23 that's called Fe2+.
00:04:22.17 And then some process, which I'll get to in just a minute, oxidized this ferrous iron to ferric iron,
00:04:29.00 and at that point it could react with constituents in the waters such as hydroxyl species,
00:04:34.26 to form iron minerals, such as this one: ferric oxyhydroxide, rust.
00:04:41.01 And over time this mineral transformed and changed
00:04:44.19 into different types of minerals, became compacted,
00:04:47.17 and mingled with others and wound up in these rocks that we today know as banded iron formations.
00:04:54.14 But this initial step here is the critical one in terms of giving us some insight into microbial
00:05:00.23 activities on the ancient Earth. And let's think about two scenarios where microorganisms
00:05:06.05 might have been involved. The first scenario
00:05:09.03 is one where a very primitive type of photosynthetic organism,
00:05:12.27 well, I should say primitive in quotes, because actually this metabolism is remarkably sophisticated.
00:05:17.16 Nonetheless, this is primitive in the sense that it is a type of photosynthesis
00:05:21.18 that does not generate oxygen. Rather it is called anoxygenic, meaning that there is
00:05:29.09 an electron donor, in this case ferrous iron, that is oxidized to ferric iron
00:05:35.29 and that powers the reduction of inorganic carbon, CO2, to biomass.
00:05:42.18 And you can see this is a very dramatic metabolism
00:05:46.25 when it occurs because all you need is light, microorganisms, and ferrous iron,
00:05:52.21 and a few other things to help them get going, but those are really the three most important ones
00:05:57.22 in a bottle here, with, as I said, a few nutrients added so they can do their thing,
00:06:04.19 and when light is shined on this bottle,
00:06:08.24 these organisms very rapidly are able to oxidize the iron.
00:06:12.19 And they produce rust, and you can see the rusty color here in this bottle.
00:06:16.14 And this rust is exactly the type of iron that is the predecessor of the minerals that constitute
00:06:22.04 these banded iron formations. Now in the middle you see these organism growing on a different
00:06:27.03 electron donor, and I'll get to what I mean by an electron donor and an electron acceptor later in this lecture.
00:06:33.17 And in this case they are utilizing hydrogen as an electron donor, and the pink color you see
00:06:39.13 is due to photosynthetic pigments in their membranes
00:06:42.16 that enable them to harvest light and grow in this way.
00:06:45.07 So this scenario, as I said, is one that is catalyzed by
00:06:51.01 organisms that do not generate oxygen. They are anoxygenic phototrophs
00:06:55.14 capable of oxidizing iron in a photosynthetically mediated
00:06:58.29 process under environments where no oxygen is present whatsoever, and yet these ferric minerals can form.
00:07:07.22 Now scenario two, that is entirely different
00:07:11.02 is one where the organisms that ultimately catalyze the precipitation
00:07:15.11 of these minerals were producers of molecular oxygen, and these are the cyanobacteria
00:07:21.24 that you can see here that were critically important in the history of the evolution of metabolism
00:07:27.08 and quite frankly also in changing the overall chemistry of the Earth including its atmosphere
00:07:33.04 because they evolved the ability, the remarkable ability, to use water as an electron donor in photosynthesis,
00:07:40.05 oxidize it to molecular oxygen, and through this process, power the reduction of CO2 to biomass.
00:07:48.19 Now once they produce this oxygen, the oxygen chemically would have been able to react
00:07:55.09 with ferrous iron, oxidizing it to ferric iron,
00:07:58.25 and then this in turn would go down the pathway to precipitate these rusty minerals I showed you.
00:08:03.06 So here we have two options: one scenario where no oxygen is involved,
00:08:10.11 and a second scenario where oxygen is mandatory.
00:08:12.21 And both of these are biological processes.
00:08:15.22 So how do we distinguish between them if we are interested in understanding
00:08:20.19 the types of organisms that were present on Earth in the remote past?
00:08:23.29 Well this is quite a challenge, indeed, and there will be many years of investigations in the future
00:08:31.07 in order to really pin this down.
00:08:32.29 And it is a great field to get into if you are a beginning student
00:08:36.07 and interested in both biochemistry and evolution,
00:08:39.16 but what I'll say just for now is that we know from a variety of indicators
00:08:43.23 that somewhere between 2 billion and 3 billion years old
00:08:47.26 it is very probable, indeed it is almost certain,
00:08:52.05 that the process of oxygenic photosynthesis arose.
00:08:54.19 But when exactly this happened and how the evolutionary events came together
00:09:00.19 such that these anoxygenic phototrophs that can utilized reduced substrates
00:09:06.05 such as hydrogen, or sulfur species, or iron as electron donors in photosynthesis
00:09:12.13 morphed into a more sophisticated type of phototroph,
00:09:18.00 that was capable of using water as an electron donor,
00:09:20.25 the cyanobacteria, which in turn, are what became the plastids,
00:09:26.22 the chloroplasts that we find in modern marine algae
00:09:30.10 and also of course, in plants that are very well known for their ability to do oxygenic photosynthesis.
00:09:36.09 We do not know. We do not know when this happened.
00:09:39.14 And in my third lecture in this series, I will discuss ways that we can begin to approach this problem.
00:09:44.22 But it's a profound question, and what I would like to leave you with now
00:09:47.27 is just the simple message that these very ancient rocks, such as these banded iron formations,
00:09:52.16 here are holding clues to a mystery that we have to unravel.
00:09:56.22 And it is through tools of modern biology that ultimately we hope to get there.
00:10:01.13 All right, now as I said the history of microbial life
00:10:06.19 extends very far back in time, as far as 3.8 billion years as we currently estimate,
00:10:12.22 but this might have been even earlier for all we know.
00:10:15.22 How do we decipher when particular microbial metabolisms evolved and what types they were?
00:10:22.04 Well, this indeed is extremely challenging.
00:10:24.09 And there are three primary ways that we can gain insight
00:10:28.04 into the microbiology of the past through using either
00:10:31.25 morphological, molecular, or genomic, which is of course a form of molecular biosignatures.
00:10:38.21 And these are very different in what they can tell us.
00:10:41.20 So the first two, morphological and molecular,
00:10:45.01 are important because they can be concretely linked to rocks, old rocks, that we can date.
00:10:51.02 And because of this when we see a particular form,
00:10:55.09 this is being held in the hand a sample of stromatolite.
00:10:58.29 This is at a very different scale here. You are looking at a thin section of a rock,
00:11:03.14 and that is true for these images below
00:11:05.28 where the scale is about 1 millimeter, in this image, and it is even smaller down here.
00:11:12.10 The structures that you observe have been interpreted as being vestiges of ancient life for various reasons.
00:11:19.13 But this interpretation is often ambiguous, and it is a challenge to be able to come up with unambiguous
00:11:25.06 biosignatures simply on the basis of their shape.
00:11:28.12 And so geobiologists, those interested in seeking to understand life in ancient times,
00:11:35.02 have turned recently to what we call molecular biosignatures that come in two forms:
00:11:39.27 either organic biosignatures, or some type of inorganic biosignature,
00:11:46.26 often expressed as a ratio of different isotopes in a sample.
00:11:51.04 Now this in turn is challenging as well,
00:11:54.25 but it may be the best way that we can gain more specific insight into
00:11:59.18 different types of metabolisms, by looking at actually the chemistry what is left in the rock
00:12:04.10 and being able to deduce through finer scale analyses whether or not
00:12:09.14 this chemistry was one that was uniquely imparted by a biological process.
00:12:13.13 Lastly we can think of genes as fossils, and the genomic record has been crucial
00:12:20.03 in establishing the diversity of life on the planet, as I'll get to in a little while in this lecture,
00:12:25.21 but it also helps us understand the relatedness of different enzymatic functions
00:12:30.22 and how they evolved from one another.
00:12:33.19 While this does not give us a concrete date when these metabolisms
00:12:37.14 evolved, it does provide us with an ability to look at
00:12:41.10 the relationship between different metabolisms, and come up with an order in which they likely were invented.
00:12:49.06 So that is all I am going to say right now on the antiquity of microbial life,
00:12:53.22 and if you are interested, tune in for lecture three in this series,
00:12:56.22 where I will spend some more detail talking about how we use a particular compound found in lipids
00:13:02.13 in modern cells as a potential indicator for oxygenic photosynthesis
00:13:06.19 and whether or not this is a valid thing to do.
00:13:10.01 The next point now I want to turn to is just how numerous microbes are.
00:13:14.22 So let's ask a very simple question. How many microbes are there on Earth?
00:13:18.11 And to bring this into a human reference point, let's begin with the number of the human population.
00:13:23.19 So I am from Los Angeles, which at the latest census,
00:13:27.27 was around 10 million people. And in the state of California, we are up to approximately 35 million,
00:13:34.11 and in the United States in general, nearly 300 million.
00:13:39.16 These are large numbers, but overall in the world we are up three orders of magnitude.
00:13:44.18 at 6 billion people. And that's a lot of folks.
00:13:47.27 However, this is nothing in comparison to the microbial population
00:13:51.18 as estimated by a wonderful paper that I am citing here at the bottom of the slide
00:13:55.22 called, "Prokaryotes, the Unseen Majority" that was published in PNAS in 1998.
00:14:01.29 These are very rough numbers, but give or take an order of magnitude
00:14:05.01 here or there, I think you are going to be impressed when you see the number that I am about to show you.
00:14:09.06 So the estimates for the microbial population are just enormous, 5 times 10 to the 30th cells.
00:14:15.08 And this indeed is such a large number that it is very difficult to wrap our minds around it.
00:14:20.21 So to try to make this a bit easier to do,
00:14:22.16 I did a very simple calculation, where I assumed that the length of a given micro-organism
00:14:27.01 was one micron and asked, "how many times would we need to go back and forth between the Earth
00:14:33.11 and the Sun if we lined up all of these organisms end to end in order to account for this number?"
00:14:38.29 And the answer, shockingly, is we would need to go back and forth 200 trillion times.
00:14:45.12 So hopefully that impresses you with just how many of these creatures there are on the planet.
00:14:49.28 Now where are they, if there are so many?
00:14:52.08 How come we don't think about this all the time?
00:14:54.27 Why aren't we overwhelmed?
00:14:56.04 Well, one reason is that oftentimes we are shockingly ignorant
00:14:59.19 about the fact that they are all around us, that we ourselves are walking micro-organisms.
00:15:04.04 So one of the first scientists to appreciate this profound fact
00:15:08.10 was the father of microscopy, Antony van Leeuwenhoek.
00:15:11.11 And this is a lovely image that he drew from his observations down his first microscope
00:15:18.01 in 1684, and you can see he drew some nice rods and cocci, and even pictures of probably motility
00:15:26.22 what is meant by these dotted lines from C to D.
00:15:30.29 And he reflected, as he was looking through the microscope about his own teeth, and this is I think a very funny quote.
00:15:37.28 He said, "Though my teeth are kept usually very clean,
00:15:40.24 nevertheless when I view them in a magnifying glass,
00:15:43.25 I find growing between them white matter as thick as a wetted flower.
00:15:47.09 The number of these animals in the scurf of a man's teeth,
00:15:50.05 are so many that I believe they exceed the number of men in a kingdom."
00:15:54.17 Well, this indeed is actually an underestimate.
00:15:58.28 Not only do they exceed the number of men and women in a kingdom,
00:16:02.13 they go far beyond that. So if we actually look at our own bodies...
00:16:06.13 just take a look at your wrist, at one square inch on the surface of your wrist.
00:16:10.23 Right there, we are estimated to have five to fifty thousand bacterial cells.
00:16:16.22 And it just increases in density as we move to other parts of the body, such as the groin and the underarms,
00:16:22.28 in our teeth, and really where it's mainly at in our bodies is in our colon.
00:16:29.06 And the overall total per person is seventy trillion.
00:16:33.25 That is quite a lot.
00:16:35.24 And one thing that I think is really important for you
00:16:38.18 to know about the microbial community within your own body,
00:16:42.16 is that there are ten times the number of microbial cells
00:16:47.04 in our system than there are human cells.
00:16:51.21 And not only that, when we look at the genetic potential of the DNA
00:16:56.09 within these organisms, the genetic potential of only those within our guts
00:17:02.15 is over one hundred times that of the human genome.
00:17:06.21 So you might begin to ask whether or not humans are not merely walking vats of microorganisms,
00:17:11.29 carriers serving their existence.
00:17:14.08 It is something to think about, and there's a great deal of research now emerging
00:17:18.20 that is beginning to illuminate just how crucial these organisms are
00:17:21.25 for human health, not only with regard to being able to help us digest our food,
00:17:27.07 but also interfacing and controlling our immune system,
00:17:30.09 in ways that are fascinating and profound.
00:17:32.29 Now despite the fact that this number, ten to the twelfth, seems really large, and indeed it is,
00:17:38.25 it's peanuts when we compare it to other domains where we find microorganisms.
00:17:44.09 So let's start with the least abundant, up in the air,
00:17:46.24 It is quite amazing to me that they've been detected as high as thirty four to forty six miles up
00:17:53.06 into the sky. But these concentrations are really small relative to other compartments.
00:18:00.00 As I told you, within the human body we have quite a few.
00:18:04.12 And when you add up all of the humans and domestic animals, and then termites,
00:18:08.08 which I'll get back to in just a bit,
00:18:09.20 the order of magnitude jumps up to about ten to the 23rd, to 24th
00:18:14.06 This is superceded by the quantities that you can find in soils,
00:18:20.06 in forests and grasslands, deserts, tundras, swamp environments.
00:18:24.10 These places are very fertile homes for microorganisms and there their activities can transform
00:18:29.23 the chemistry of their environment quite profoundly.
00:18:31.29 And this is of course also true in aquatic domains, where at similar orders of magnitude
00:18:38.08 we find microorganisms in both marine and freshwater environments.
00:18:42.01 But all of these numbers pale in comparison to the numbers that we find in the subsurface,
00:18:46.14 both in terrestrial and oceanic environments, where microorganisms
00:18:51.09 have been detected as deep as two miles.
00:18:54.22 Now, this really is a very interesting frontier area in microbiology
00:18:58.05 It is hard to go down into these depths, and yet nowadays, researchers are equipped with the tools
00:19:05.02 they need in order to access these remote communities.
00:19:07.26 And what remains to be learned is what exactly these organisms are doing in situ.
00:19:12.11 Are they active? And if so, what are their activities?
00:19:15.26 Are these activities affecting in a significant way
00:19:19.05 the physical and chemical properties of these environments?
00:19:22.07 We don't know, and we look forward in the coming decades to finding
00:19:25.04 the answers to these and other interesting questions.
00:19:27.19 So now let me just give you an example, a tour through various parts of the world
00:19:34.05 and other inhabitants of that world where we find these organisms.
00:19:39.17 Just to bring home to you how ubiquitous microbes are on the planet.
00:19:44.07 So to start with what might be a more familiar image, here what you are looking at is pond scum.
00:19:51.09 You are looking at a wonderful assemblage of phototrophs
00:19:54.27 and other microorganisms in this pond. And my favorites of course are these purple phototrophs.
00:20:00.03 These are the ones that I told you about earlier that are what we call the anoxygenic phototrophs
00:20:04.27 that are not utilizing water as a substrate in photosynthesis,
00:20:08.29 but are utilizing other more reduced compounds such as different types of sulfur species, hydrogen, or iron.
00:20:17.15 Now these organisms that we see in modern day ponds, as I told you at the beginning when I was illustrating
00:20:23.20 the antiquity of microbial life with the example of the banded iron formations,
00:20:27.25 are absolutely historically important for their metabolism,
00:20:33.19 and the diversity of their metabolism, and how it's changed the geochemistry of the Earth.
00:20:38.00 Not only has the evolution of photosynthesis contributed to evolving our atmosphere
00:20:43.20 to one that contains oxygen over the course of time,
00:20:46.14 but as I also showed you with the banded iron formations,
00:20:48.29 these types of organisms have likely shaped ore formation as well.
00:20:54.21 And many other important processes have been able to come about
00:21:00.21 thanks to these organisms doing what they do, and it should be noted
00:21:05.15 that this type of metabolic activity, photosynthesis, is one that today we are highly interested in
00:21:12.06 because of our need for coming up with alternative energy sources, and
00:21:16.25 certainly if chemists were able to mimic what these wonderful microbes in this pond do, we would
00:21:22.25 be able to not worry so much about our dependence on foreign oil
00:21:27.02 and our fossil fuel supplies being burnt, but that's a story for another day.
00:21:32.08 The point is, their metabolic diversity is old. We see it all around us, and the biochemistry is really quite fascinating.
00:21:39.18 Continuing on with the chemistry and the metabolism of these organisms
00:21:44.28 not only do they do important things when they are growing,
00:21:48.00 but they also do important things when they start hitting what we call stationary phase.
00:21:51.24 And this is a point in their development where they're not necessarily actively growing,
00:21:56.12 but they are at a higher density and they are just hanging out metabolically.
00:22:00.26 And when this occurs in their lifecycle,
00:22:04.09 sometimes metabolites and pigments begin to be excreted.
00:22:08.08 And these pigments, which are called secondary metabolites,
00:22:12.14 although that name itself may be a bit misleading, because they are only secondary in a temporal sense,
00:22:18.14 in that they are made after a phase of active growth,
00:22:21.21 but by no means are they secondary in terms of the physiology of the organisms that produce them.
00:22:27.00 None the less, these metabolites oftentimes are used today by pharmaceutical companies
00:22:32.14 as natural products that confer antibiotic activity.
00:22:35.13 And a terrific example of this are organisms in the Streptomycetes family that you see here
00:22:40.18 in this Petri dish that are producing a whole host of wonderful antibiotic compounds.
00:22:45.02 Now containing in the environment of the soil of course are roots of plants.
00:22:51.00 And in this part of soil known as the rhizosphere,
00:22:55.04 we can find microorganisms as well that are colonizing in a very beneficial way the plant roots.
00:23:00.28 And here is a tomato root seedling. This is an image taken by Guido Bloemberg.
00:23:06.25 And he showed in experiments in the laboratory that when he took tomato root seedlings
00:23:12.27 and mixed them with an organism called Pseudomonas,
00:23:16.01 that this bacterium was able to colonize the plant and form what we call biofilms on the surface of the root.
00:23:22.18 And this is just one example of organisms that interact with plants.
00:23:26.20 There are many that fall into this category with different names.
00:23:30.12 And the bottom line is that they have a very beneficial relationship with these plants,
00:23:34.11 where sometimes they produce natural products that fend the plant off from fungal predators
00:23:40.05 and so they serve as biocontrol agents.
00:23:42.24 Other times these organisms are capable of fixing molecular nitrogen
00:23:48.14 into a usable form and essentially acting as a natural fertilizer.
00:23:53.14 Now crawling around in not only soil environments,
00:23:58.00 but of course we are very familiar with these from our homes, are termites.
00:24:01.00 And the termites are a terrific source of microbial diversity
00:24:05.17 and one that is becoming an increasingly important micro-environment
00:24:10.07 in which to look because of our desire to understand microbial processes
00:24:16.00 that might be harnessed for lignocellulose degradation.
00:24:19.04 Again, out of a need to develop alternative sources of energy.
00:24:22.22 Now a colleague of mine, Professor Jared Leadbetter at Caltech
00:24:26.14 studies these termites, and he likes to call them "an ecosystem in a microliter".
00:24:31.05 And I think this is really a fantastic description of them because it is within their hindgut that you find a zoo
00:24:38.21 of microorganisms and protozoa that are swimming around
00:24:43.12 doing all sorts of important activities that make it possible for the termites
00:24:47.12 to digest their wood. And in the process they emit methane,
00:24:52.02 and not an insignificant fraction of this methane ultimately makes its way
00:24:55.16 up into the atmosphere and contributes to the overall chemistry on the planet.
00:25:02.17 So speaking of methanogens, here you see
00:25:05.08 a dramatic illustration of them at work.
00:25:07.18 This image that I am standing in front of is taken from Cedar Swamp in Woods Hole, Massachusetts.
00:25:12.15 And it is an image from a group of students from the microbial diversity class,
00:25:18.02 which is a fantastic course for about twenty students, half from the United States and half from overseas,
00:25:24.07 who come together every summer to understand how
00:25:27.00 microorganisms are able to perform these various metabolic activities
00:25:32.08 that I have been describing in these lectures.
00:25:34.26 And what you can see here is that the students have gone waist deep into this swamp
00:25:39.19 and they have stomped around, and as they have done this they have collected the bubbles that come up
00:25:46.03 as they stomp the sediment, and collected them in these inverted funnels.
00:25:51.11 And then some brave individual holds that funnel
00:25:54.14 and removes their hand just at the moment when a friend comes by with a flame,
00:25:59.09 and ignites it, and here you see a lovely illustration of methane at work.
00:26:04.25 So methanogenesis led to the creation of the methane gas that was ignited here.
00:26:10.06 Now in the past the activity of these organisms that generate this methane
00:26:15.23 that are called methanogens, might have been important in shaping the chemistry of the Earth's environment.
00:26:19.27 And the reason we suspect this may be the case is because early in Earth's history the environment
00:26:25.00 contained appreciably more methane than it does today.
00:26:28.18 Now a different example of a habitat where microorganisms are very important
00:26:33.21 is in Chile and in other places on Earth,
00:26:37.19 but this example here is taken from the Andina Copper mine in the Andes in Chile
00:26:44.23 where microorganisms are exploited for their abilities to help
00:26:48.27 with bioleaching. And so what happens is that in these mines there are piles
00:26:54.06 that are built up, and they are fertilized essentially with indigenous microbial populations
00:27:01.08 that are able to live in shockingly low pH levels,
00:27:05.17 down to pH as low as one, and sometimes even lower.
00:27:08.23 And these organisms are essentially eating the minerals in this mine pile
00:27:14.14 and the process of metabolizing it, changing the mineralogy, in such a way
00:27:19.06 that copper is solubilized and leached. So here is another example of an environment
00:27:25.27 that is quite extraordinary and yet microorganisms have been able to adapt and even to thrive in this extreme condition.
00:27:33.04 So on our tour of extreme pHs, we just saw an example of low pH, so let's go to a high pH environment.
00:27:39.24 This one I am showing you is Mono Lake that is in Northern California.
00:27:44.09 And Mono Lake is quite an extraordinary place. It looks almost like it is from another planet.
00:27:49.25 You see these beautiful tufa towers that are calcium carbonate minerals forming,
00:27:55.05 and it is because the pH is so high and the alkalinity is so high that they naturally precipitate from these waters.
00:28:01.19 In addition to having these carbonate minerals, contained within this lake environment
00:28:07.09 is a ton of arsenic, and I will get to this in part two of my lecture today.
00:28:11.07 And what I want to point out right now is that in this very high pH environment, and also one
00:28:17.14 that's replete with arsenic, nevertheless we find organisms called alkaliphiles
00:28:21.23 that thrive here, that are able to make a living utilizing arsenic as a terminal electron receptor in respiration.
00:28:30.05 This is the subject of my second lecture. And in so doing account for 14% of the carbon turnover in this system.
00:28:38.23 Now let's go on to another example of an extreme environment.
00:28:42.14 Here now we are looking at an extreme of salt.
00:28:45.26 And there is no better example of this than the Dead Sea in Israel, but
00:28:50.06 you can find organisms such as those that inhabit the Dead Sea also in the Great Salt Lake,
00:28:56.04 and other places on Earth such as salt flats, where you have very high salt content.
00:29:04.19 And the organisms living here are capable of growing despite this high salt
00:29:08.21 and have adapted particular molecular strategies to cope with it.
00:29:12.15 One very elegant example of this is their ability to use special photopigments called rhodopsin
00:29:18.23 and these are colored purple.
00:29:21.24 And these rhodopsins, they have in their membranes, and enable them to generate energy
00:29:27.11 under conditions where they need to use slightly different
00:29:30.02 strategies than organisms that are growing under
00:29:33.28 conditions that we would consider more normal.
00:29:36.27 Now, so approaching the end of our tour through microbial diversity and ubiquity, I want to end with a few other extremes
00:29:44.00 now that are based on temperature and pressure.
00:29:46.04 If we think about the extremes of cold there is no better place to go than Antarctica.
00:29:50.27 And you might be surprised to realize that even in this environment you have microorganisms thriving in the crust.
00:29:57.02 And these organisms are psychrophiles, and they're ability to grow is dependent upon dust
00:30:02.26 from winds carrying nutrients picked up from the continents surrounding Antarctica, South America, Australia, Africa,
00:30:14.24 that reach Antarctica, deposit their dust and fertilize these upper crusts of the ice
00:30:20.14 where we have intrepid pioneer organisms that are able to utilize these nutrients
00:30:26.03 and grow, even in these very cold regimes.
00:30:28.02 So another extreme is that of temperature and pressure, and there is no better environment in which to observe this
00:30:34.24 than at the bottom of the ocean, in environments where we have hydrothermal vents
00:30:39.21 that are releasing nutrients into the deep. And here is an example of one of these vents. It is called a black smoker
00:30:46.17 because the nutrients that it releases, including manganese and iron,
00:30:51.14 often precipitate in the conditions of the oceans at these sites
00:30:57.16 such that they look black.
00:30:59.22 Now around these vents there is abundant life,
00:31:04.07 really extraordinary life, not just microbial life.
00:31:06.08 but giant tube worms, and fish, and other macroscopic organisms.
00:31:10.10 So the ability of all of this abundant life to be in this environment
00:31:15.10 crucially depends upon the activities of microorganisms that are chemosynthetic.
00:31:20.01 that are able to grow by the oxidation of sulfur and other compounds that you have
00:31:25.03 present in this environment, and couple that oxidation of these reduced substrates to
00:31:31.05 the fixation of CO2 into biomass.
00:31:34.15 And this is at the base of the food chain that then sustains the growth of other marine organisms.
00:31:40.12 such as these tube worms. And here you see an example of that
00:31:43.10 in these beautiful tubeworms. If you cut them open and you look at one of their organs,
00:31:50.29 called the trophosome within these organs are bacterial symbionts
00:31:55.27 that are doing the process that I just mentioned.
00:31:58.08 So my final example that I will end with
00:32:01.12 is one that might be the most familiar to you
00:32:04.02 if you have ever done any PCR in molecular biology.
00:32:06.25 So most of you have heard of the enzyme Taq polymerase,
00:32:10.06 and this polymerase is what allows us to do an amplification reaction when we are doing PCR.
00:32:18.21 Now this enzyme, Taq, derives from a bacterium called Thermus aquaticus,
00:32:24.28 that is where the Taq comes from. The "T" is from the Thermus and the "aq" from aquaticus.
00:32:31.05 And this is a thermophile that was isolated in Yellowstone
00:32:35.14 at a hot spring, many decades ago.
00:32:38.03 And it was presciently realized by Kary Mullis
00:32:41.18 and others that the enzymes contained within it could be useful for various biotechnological applications
00:32:47.26 because they wouldn't denature at the temperatures that would kill most other types of cells.
00:32:54.02 So these thermophiles are a very fascinating group of organisms
00:32:57.28 whose molecular adaptations include not only DNA polymerases,
00:33:03.26 but also a wide variety of other enzymes that might be of industrial use.
00:33:10.01 So let's now end with diversity, which is really my favorite part
00:33:14.14 of the microbial world. And I want to cover a few different areas of this.
00:33:18.18 The first is phylogenetic diversity.
00:33:20.03 Now one of the most important lessons to be learned in evolutionary theory
00:33:25.03 was learned several decades ago from work by Carl Woese and his colleagues,
00:33:30.03 including Norman Pace, who applied Carl Woese's fundamental
00:33:34.07 insights into the diversity of life to the natural world.
00:33:38.02 And these individuals together with others were able to demonstrate very clearly
00:33:44.05 that when we think about the diversity of life out there on the planet,
00:33:48.16 we are really talking about a microbial world, whether we call these
00:33:52.22 microorganisms Bacteria or Archaea or even Eucaryotes.
00:33:58.23 What I want you to appreciate is that when you look at the tree of life,
00:34:03.11 that's what this is. It is a tree that is drawn based upon comparing the sequences of a very particular molecule
00:34:11.06 that every living organism has, that is ribosomal RNA, that is necessary for the process of translating
00:34:19.07 messenger RNA into protein.
00:34:21.07 Because this is a very universal and highly conserved molecule
00:34:24.28 Carl Woese and colleagues were able to deduce that it was a beautiful molecular chronometer
00:34:31.01 that we can employ to look at the evolutionary relatedness between different organisms.
00:34:36.16 And when he and his colleagues did this , he recognized that
00:34:39.19 there were three primary domains of life, the Bacteria, the Archaea, and the Eucarya.
00:34:43.16 And moreover, what I want to stress now is that our entire universe of Homo sapiens
00:34:52.12 and humans and plants and animals, the macroscopic eucaryotic world,
00:34:57.12 is only occupying in terms of this space on the tree,
00:35:01.27 which is known as a phylogenetic tree, meaning a tree of evolutionary distances
00:35:06.21 between different types of life forms, a very tiny miniscule branch.
00:35:12.00 And everything else that I am showing here is microbial.
00:35:14.13 So hopefully that impresses you, but before we leave this tree let me point out two more
00:35:19.20 facts that are very important.
00:35:20.29 All of the metabolism on the planet was invented by microorganisms
00:35:26.05 including the metabolism that we perform in our bodies today in our mitochondria.
00:35:31.11 So the mitochondrion is nothing more than an ancient bacterial cell
00:35:36.28 that invented the ability to do oxidative phosphorylation, which I'll tell you about in a little bit,
00:35:42.24 that was engulfed or brought into symbiosis with some other type of cell,
00:35:47.23 and over the course of time involved into the organelle that we call the mitochondrion.
00:35:52.28 But it was a microorganism first, and that is where the beautiful metabolism that it goes through was generated.
00:36:02.14 The same story is true for the chloroplast. This is nothing more
00:36:06.03 than cyanobacteria that over time turned into plastids and became incorporated into other cells.
00:36:11.25 Now the next important point I want to make is that microbial diversity also manifests itself morphologically.
00:36:18.19 And this is something that only recently we are coming to appreciate in its full glory.
00:36:24.02 Back in the days of Leeuwenhoek, when he had a simple microscope, all he could really see were different shapes of microbes,
00:36:30.24 and to be quite honest, that is not terribly spectacular and includes rods and spirals and some cocci.
00:36:37.11 Once in a while you see higher structures forming of communities however,
00:36:41.25 and Leeuwenhouk didn't necessarily know about these,
00:36:44.05 but here is an example of one here. This is a beautiful example
00:36:48.13 of fruiting bodies beginning to form by the soil organism Myxobacteria
00:36:53.10 that does all sorts of interesting things when it comes together in a group
00:36:57.01 that it wouldn't do as any individual cell.
00:37:00.25 This is social behavior. So this is an example of bacteria acting in a multicellular fashion, if you will.
00:37:06.11 Microorganisms, however, can get remarkably large. They are not just on the scale of microns.
00:37:11.21 And here is a good example of this.
00:37:13.20 This is, to my knowledge, one of the largest microbial
00:37:16.28 cells known to date. It is called Thiomargarita namibiensis,
00:37:19.29 which means the sulfur pearl of Namibia.
00:37:22.17 And it is on the same scale as the eye of a fruit fly.
00:37:25.13 And when you look at it in more detail, the reason it is so big is that it contains this huge vacuole
00:37:31.21 filled inside with nitrate, which is one of the substrates it uses to power its metabolism.
00:37:38.02 And it couples the reduction of nitrate to a more reduced form of nitrogen
00:37:43.07 to the oxidation of sulfide, and in this way it powers energy for growth.
00:37:48.00 But let's leave the metabolism aside and stay focused now just on the form.
00:37:52.03 Here is an example of one of my favorite organisms, Rhodopseudonomas palustris,
00:37:57.02 and the reason I am showing you this is simply to illustrate
00:37:59.24 that it has quite an amazing membrane structure within it.
00:38:05.27 One that is reminiscent even of the Golgi in higher organisms.
00:38:09.09 And indeed it might have been the progenitor of that at the cell biological level.
00:38:14.17 And how these various structures form, these are what we call
00:38:18.06 the inner cytoplasmic membranes where the photosynthetic machinery is housed in this case,
00:38:22.19 in terms of the detail of what creates their shape is an open and exciting question
00:38:27.08 that future microbial cell biologists will no doubt solve.
00:38:30.17 But the final example, which is probably my all time favorite, is of an organism called a magnetotactic bacterium.
00:38:38.16 And here you see if you just look at it in a light microscope,
00:38:41.15 although this is actually an image of fluorescence where we have put some GFP into the bug,
00:38:46.04 it looks like just a common spiral.
00:38:48.09 If you take a fancier microscope, a transmission electron micrograph, and cut it
00:38:53.01 open and do a thin section, you can see that it has this beautiful chain of magnetic particles inside it.
00:39:00.00 And now what I am going to show you is, I think, the best advertisement for the beauty of bacterial cell biology
00:39:05.28 that I know, and it is a cryo-electron tomogram of one cell.
00:39:13.10 And this was work done by Arash Komeili who is now a professor at UC Berkeley
00:39:18.26 and his collaborator Zhuo Li in Grant Jensen's lab at Caltech. And together
00:39:23.17 we made this movie showing the internal structure of these organisms.
00:39:27.26 So what you are going to see now is coming up through the bacteria different sections,
00:39:32.11 and here you see the magnetosomes coming into view. Those are the membranes that contain the magnetite.
00:39:39.10 If you missed them, now look, OK.
00:39:41.02 Here they are in red, those magnetosome membranes, and then there is this yellow filament surrounding them.
00:39:46.27 And what we have come to appreciate is that this filament is a protein that is very similar to actin.
00:39:52.11 And it is necessary for these magnetosomes, for these organelle-like,
00:39:58.01 although they never separate from the membrane, so they are not true organelles.
00:40:01.14 Here you see they're attached by a neck that's only 5 nanometers in diameter, which is quite amazing, to this inner membrane.
00:40:10.24 They invaginate and form these vesicles within which a beautiful single domain crystal of magnetite can form.
00:40:19.19 And this order, the fact that they are linear in a chain,
00:40:22.21 is enabled by a cytoskeletal filament, an actin-like protein.
00:40:28.17 OK. So the next to the last point that I want to make on diversity
00:40:33.09 is behavioral diversity, and there is another lecture in this iBioSeminar series
00:40:37.06 by Professor Bonnie Bassler from Princeton that can give you more information about this if you are interested.
00:40:41.28 But what I wanted to point out here while we are going through a tour through diversity
00:40:46.13 is simply that microorganisms can act in ways that are quite extraordinary
00:40:51.15 when they are acting as a group.
00:40:53.11 And you can see that illustrated by the activities of the bacterium Vibrio fischeri within the light organ of a squid.
00:41:01.10 And here is an image that is from the beautiful pioneering work of Margaret Mefal-Ngai
00:41:06.18 and Ned Ruby at the University of Wisconsin, Madison where they have been studying for decades the interactions
00:41:12.01 between the microorganisms in the light organ of the squid and the squid,
00:41:16.28 and the ability of these organisms to colonize this environment,
00:41:20.06 and when the lights go out at night, emit a beautiful luminescence. Here you can see pictures of these organisms
00:41:28.12 that have just been streaked out on a plate in the dark.
00:41:30.11 They are glowing. Well, they glow as well here at night in the belly of the squid.
00:41:35.27 And it shields these squids from predators below
00:41:39.00 because the light of the moonlight coming down from the top
00:41:41.28 is roughly of the same luminescence as the light that they are emitting.
00:41:45.26 So it allows them to have a stealth function and glide around in the oceans
00:41:50.19 and be unseen to predators deeper below them.
00:41:56.07 Now, this isn't just a phenomenon that affects the squid. This is a phenomenon that can get quite enormous in its scope.
00:42:03.19 And the best example to illustrate this is this satellite image here taken off of the Somalian coast,
00:42:09.11 where you see an image that quite literally is of milky seas as described by the ancient mariners,
00:42:17.21 but what today we understand as glowing bacteria.
00:42:21.26 In this case an organism, likely called Vibrio harveyi, associating with micro-algae in this environment
00:42:28.22 that for whatever reasons that are not fully understood at this particular point in time
00:42:33.22 when this satellite image was taken, had a bloom and began luminescing like crazy, and filled up a volume the size of Connecticut.
00:42:41.27 All right, so, to end I want to just mention a few rules of microbial diversity
00:42:47.23 because almost everything that I have talked about so far
00:42:51.11 in this lecture ultimately comes back to the ability of organisms to generate energy in ways that are quite amazing.
00:42:59.06 And I would stipulate that microbes are by far the best chemists on the planet.
00:43:02.25 And so if you are a chemist, pay attention, because a lot of lessons can be learned from these guys.
00:43:07.06 All right. Now when we are talking about the phase of active growth,
00:43:11.13 the bottom-line that microbes are facing is they simply want to divide.
00:43:15.12 And to do this they need two things. They need energy, and they need carbon.
00:43:20.08 And beyond that, they are virtually unconstrained, although there are a few constraints,
00:43:26.25 and we will come back to that in a moment.
00:43:29.03 They need substrates, and these substrates can be organic or inorganic compounds.
00:43:36.11 This is now for the part where they are going to be generating energy.
00:43:40.22 Those substrates are converted to products through catabolic reactions, or energy generation, if you will.
00:43:49.09 And often times we think of energy generation in the form of ATP,
00:43:52.20 the most important energy carrying molecule within the cell.
00:43:57.06 Now this part of metabolism, catabolism, is coupled to anabolism,
00:44:03.12 which is the part of metabolism that is concerned with energy consumption, or biosynthesis.
00:44:08.12 And now down here what we are talking about is the conversion of
00:44:12.01 carbon, often in the monomeric form, to biomolecules that are far more complex, so protein, DNA, lipid, for example.
00:44:22.27 Now if we are thinking just about the substrates, as I said they can come from a variety of sources.
00:44:30.06 Always they're chemical, although light can help enable cells
00:44:34.21 to actually utilize those chemicals in ways that they otherwise wouldn't be able to do.
00:44:39.10 But when we are talking about the growth of organisms just purely on chemicals, without needing
00:44:46.15 a boost from light, the name we give to this metabolism is chemotrophy.
00:44:50.18 And that in turn is classified into two different types, inorganic and organic.
00:44:57.06 And when we are talking about inorganic sources of energy like hydrogen, and sulfide, and iron minerals,
00:45:03.18 this is called chemolithotrophy. And when we are talking about growing on organic substrates like glucose, or glycerol, or acetate,
00:45:11.23 this is called chemoorganotrophy.
00:45:14.04 And of course, as I said, while chemistry is always at the basis for any type of metabolism,
00:45:20.20 there is a photochemical boost that is often necessary,
00:45:25.09 when activating a compound that otherwise might not be biologically utilizable
00:45:30.16 for energy, and that is when we call that process a phototrophic one.
00:45:36.10 So the final part of this that I want to just mention is that the carbon source,
00:45:42.02 which is distinct or can be distinct from the energy source...
00:45:44.28 sometimes they are the same thing, but they don't have to be the same thing...
00:45:47.26 is either coming from inorganic carbon, CO2, or organic carbon.
00:45:52.19 And when it is coming from inorganic carbon that is called autotrophy,
00:45:55.21 and when it is coming from organic carbon, that is called heterotrophy.
00:45:58.22 So we are heterotrophs, we need to eat some type of organic carbon whether we are vegetarians
00:46:04.28 or meat eaters, but microorganisms are far more sophisticated.
00:46:09.12 They can eat minerals. They can just take CO2 from the air, and they'll be on their way.
00:46:17.14 So finally the last part I want to mention about metabolic diversity writ quite large
00:46:22.29 is that you can generate ATP through one of two different ways.
00:46:26.13 The first way is through what is called substrate level phosphorylation.
00:46:29.15 And this is also termed fermentation, and essentially is the process
00:46:33.22 where different types of reactions between chemicals within a cell
00:46:39.26 enable transfer of an inorganic phosphate ultimately to ADP to produce ATP.
00:46:49.21 And this process is enabled by chemical rearrangements within the cell
00:46:55.05 and reactions one on one between compounds.
00:46:57.18 The next major way that ATP can be formed in a cell is through the remarkable process of oxidative phosphorylation.
00:47:03.10 Basically this is about electron transport chains in membranes
00:47:07.23 that are coupled to generating a battery around a membrane
00:47:11.08 by extruding protons to one side and polarizing it so that there is an electrochemical potential gradient
00:47:18.00 across this membrane that can be harnessed to do the work of making ATP.
00:47:22.06 Now something that I am not showing you on this diagram,
00:47:25.03 but I want to introduce as terms are an electron donor and an electron acceptor.
00:47:31.00 So in metabolism there is always a substrate that is used as the primary electron donor
00:47:36.14 that can be metabolized through various pathways
00:47:39.08 and reduced to a compound that can donate electrons
00:47:43.17 to the electron transport chain in the membrane.
00:47:45.20 And then there is always something that serves as the acceptor of those electrons at the end of the chain,
00:47:52.10 and that is called the terminal electron acceptor.
00:47:54.28 And it is the path of electron transfer and proton translocation
00:47:58.22 between this electron donor and this terminal electron acceptor that is really harnessed by the membrane to do work.
00:48:05.05 And so that is what you see here pictured very generically without a whole lot of detail
00:48:10.08 in the sense that through this electron transport process,
00:48:13.20 which imagine if you will, is coupled as I said to proton translocation,
00:48:18.00 and that is achieved by different things within this membrane.
00:48:22.15 They can be proteins, or small molecules that are able to simultaneously
00:48:26.25 pass electrons through the membrane to something else
00:48:29.24 in the electron transport chain and push protons, or translocate protons
00:48:34.15 across the membrane so that there is this gradient that arises where there is more positive charge on the outside
00:48:42.07 than on the inside. Now once this happens this gradient can be used to drive ATP synthesis.
00:48:49.21 And this happens through a really amazing molecular machine called the ATP synthase,
00:48:54.09 which allows the traversal through the membrane of a proton,
00:48:58.27 that concomitantly gives the energy to phosphorylate ADP, adding that inorganic phosphate on, and making ATP.
00:49:09.08 And as this happens, the electrochemical potential gradient lessens.
00:49:15.00 And so that is what I am showing here: the energized membrane
00:49:16.25 due to proton transport coupled to electron transfer through the membrane,
00:49:22.10 and then this being expended and used in order to drive ATP synthesis.
00:49:26.12 Now while you can imagine a whole variety of things that can be electron donors
00:49:32.02 and electron acceptors from microbial metabolism,
00:49:34.27 metabolic diversity does have to conform to some rules.
00:49:37.06 And there are three that I want to point out that I think are particularly important.
00:49:41.02 The first is that the amount of energy has to be at a very minimal level,
00:49:47.14 at least in order to sustain the cell, both with regard to active growth, where you need a high level of energy,
00:49:53.27 at some threshold amount in order to double, but also at the level where you are generating enough energy simply to maintain
00:50:01.18 basic cellular processes even if they are not coupled directly to growth.
00:50:05.11 Now what is this number and how do we constrain it?
00:50:09.13 Thermodynamically, this can be expressed in this very straight forward equation here,
00:50:14.20 which is saying that the standard free energy that can be gained
00:50:18.04 from a process where there is electron transfer between the electron donor and the electron acceptor
00:50:23.13 is a function of the number of electrons transferred,
00:50:26.22 multiplied by the Faraday constant, and this in turn multiplied by
00:50:32.21 the difference in redox potential between the electron donor and the electron acceptor.
00:50:37.21 So for example, a common intracellular reductant, is NADH,
00:50:43.09 and in its oxidized state this is NAD+.
00:50:45.10 The redox potential of this redox pair is very low.
00:50:51.07 It is very negative on the electron potential scale that is typically expressed in millivolts.
00:50:57.07 On the other side of this scale are the electron acceptors with very high redox potentials, like oxygen.
00:51:04.20 And so when you couple through the membrane
00:51:09.27 a process of electron transfer from NADH to oxygen, thermodynamically you have the potential to generate
00:51:16.16 a lot of energy, and this is captured through a beautiful sequence of proton carriers and electron
00:51:24.14 transfer biomolecules contained within these membranes.
00:51:28.28 But these electron transport chains need not be between NADH and oxygen,
00:51:33.26 you can have a whole assemblage of things that can interrelate and so the minimum amount of energy
00:51:39.13 that needs to be supplied has been calculated. And this is a very crude estimation,
00:51:45.03 but it is an interesting study, and I refer you to below where you can see the reference.
00:51:49.05 Where for organisms operating in very low energy regime,
00:51:55.03 it was inferred that the minimum free energy required to sustain them and their growth
00:52:00.29 was about -4 kilojoules per mole, and that is about as low as you can go at least as experimentally measured.
00:52:06.21 Finally, regardless of the thermodynamic potential there are two other very important factors to keep in mind.
00:52:14.02 The second point is that the substrates themselves must be bioavailable. And so this is more of a kinetic problem
00:52:21.07 where we need to consider accessibility and transport of substrates
00:52:25.17 across the membrane to the site in the cell where they are used.
00:52:28.21 Or the ability of the cell to figure out a way to access them even if they can't transport them inside.
00:52:34.05 And the final point is that these substrates or the products after the metabolism has done its thing
00:52:42.00 must not themselves be toxic. So in the next couple of sections of this lecture,
00:52:47.26 I am going to give you examples of different microbial metabolisms to illustrate
00:52:52.12 these general points I have been making,
00:52:55.02 but I hope what you will remember from this seminar is the four big points about microbial diversity.
00:53:01.03 One, that it is incredibly ancient and over this long period of Earth history
00:53:06.08 numerous microorganisms in ubiquitous environments
00:53:09.18 have evolved diverse metabolisms that allow them
00:53:13.00 to catalyze fascinating chemical reactions and that these reactions
00:53:16.23 have affected not only the ability of the cells to grow and divide,
00:53:20.07 but in many instances have profoundly affected their environment,
00:53:24.09 be that environment one in an ancient ocean,
00:53:27.05 or today inside the human body.
00:53:30.18 Thank you.

00:00:00.00 Hi, and welcome back to iBioSeminars. My name is Dianne Newman,
00:00:04.09 and I am a professor at the California Institute of Technology
00:00:06.29 in the divisions of Biology and Geological and Planetary Sciences.
00:00:10.21 I am also an investigator of the Howard Hughes Medical Institute.
00:00:13.25 This is part three of my three part series in microbial diversity and evolution.
00:00:19.03 And what I would like to give you an example of today
00:00:21.19 is ways that we can through laboratory experiments
00:00:24.27 today gain insight into metabolisms that were evolved in the remote past.
00:00:31.02 And this is a very difficult task and we are just at the beginning of our ability to do it, but I hope through this story
00:00:37.25 I am going to illustrate just how interesting this field of research is and where the important open questions still lie.
00:00:44.08 So the specific example that I am going to tell you about is how we have gained some insight into
00:00:50.12 an important class of biomarkers that are used to record the evolution of oxygenic photosynthesis,
00:00:57.14 one of the most important metabolisms ever to have been invented on the Earth.
00:01:01.06 And today we see examples of oxygenic photosynthesis at work on the globe,
00:01:07.03 as you can see in this beautiful image that is taken from satellite data averaged over three years.
00:01:12.25 And what it is showing you are chlorophyll profiles in the marine oceans.
00:01:17.15 Here you see blooms of phytoplankton that occur in not only the Atlantic, but also
00:01:24.23 here in the Eastern Equatorial Pacific off of South America, and extending far out into the Pacific Ocean.
00:01:35.14 Now these gyres occur when there are blooms of phytoplankton which today
00:01:40.27 are responsible for emitting into the atmosphere most of the oxygen that we breathe.
00:01:47.16 However, these organisms, these marine phytoplankton, are not the ancestors of photosynthesis.
00:01:54.15 They are the ones that are doing it en masse in the world's oceans today,
00:01:57.27 but the process of photosynthesis evolved many billions of years ago on the Earth.
00:02:03.19 And its story is one that we still don't fully understand.
00:02:07.25 One of the most basic questions we don't understand is when did it occur.
00:02:11.09 How did organisms figure out how to use water as a substrate for photosynthesis?
00:02:15.24 One of the things we do know is that this ability to utilize water
00:02:21.27 was evolved through a complex combination of photosystems
00:02:26.16 in different types of primitive photosynthetic organisms,
00:02:30.10 including anoxygenic bacteria like the type shown in these bottles that are called purple bacteria
00:02:36.00 by virtue of the beautiful purple pigments they contain within their membranes, that you can sometimes see
00:02:41.29 when you grow them on certain substrates.
00:02:44.01 And I spoke about these organisms in my introductory talk.
00:02:47.17 And as I said then, at some point the molecular machinery
00:02:51.13 in these organisms was combined with the molecular machinery
00:02:55.12 in other organisms, also of this anoxygenic general class
00:03:00.08 but nevertheless quite distinct organisms.
00:03:02.14 How that happened, we don't know, but an issue right now is that when
00:03:09.00 this did eventually happen, and these beautiful molecular machines came together
00:03:13.24 so as to enable these organisms to utilize water as the energy together of course the real energy source is the sun,
00:03:22.24 but water is the substrate that is then activated by sunlight
00:03:27.06 to a form that is biologically utilizable in the electron transport chain.
00:03:31.19 This is when oxygenic photosynthesis was invented,
00:03:36.02 and the perpetrators of this invention were the cyanobacteria,
00:03:40.07 shown here in this slide of beautiful bubbling tubes
00:03:43.13 where CO2 is being fed into these cultures and these beautiful green organisms
00:03:48.10 with their green chlorophyll pigments are converting that CO2 to
00:03:53.21 biomass coupled to the oxidation of water to molecular oxygen.
00:03:58.16 Now the chloroplast itself as I explained in the first lecture is what evolved into the plastids that today we find
00:04:07.07 in diatoms that you see an example here. These are marine phytoplankton important in the oceans.
00:04:13.11 And of course, terrestrial plants that we are all very well familiar with
00:04:17.22 due to their ability to take water and generate oxygen.
00:04:20.10 But when this group of organisms first arose is a subject of real debate.
00:04:27.29 And it had been thought that there were signs in old rocks,
00:04:33.08 either these banded iron formations, or other geochemical signatures
00:04:36.19 that indicated oxygen likely was rising up in the oceans, being produced by these
00:04:44.26 ancient cyanobacteria, reacting with iron and precipitating it from solution,
00:04:49.11 to form these deposits, and then eventually getting into the atmosphere so that
00:04:53.17 other types of geochemical signatures found in rocks such as mass independent fractionations
00:04:59.17 of sulfur isotopes that we find in different types of sulfur bearing minerals
00:05:05.13 giving a signature that we think is very diagnostic of having oxygen present in the atmosphere.
00:05:14.08 That all of this was occurring somewhere in the middle of this time span, and certainly by around 2.4 billion years ago.
00:05:22.12 But when the ability of organisms to first take water arose, before it had this profound global effect,
00:05:30.09 is something that has not been very well constrained.
00:05:33.04 So a dominant approach that has been used to try to date the rise of cyanobacteria in the rock record
00:05:39.05 has revolved around using these organic molecules shown here that are called hopanoids.
00:05:44.04 And these are very complex organic structures that resemble sterols in eukaryotic cells.
00:05:49.19 Now the story I want tell today is a reexamination of these structures as biomarkers
00:05:56.16 to ask whether or not they are appropriate to assigning the rise of this very crucial metabolism that transformed the Earth.
00:06:05.10 So let's talk a minute about what it means to be a biomarker.
00:06:08.24 This is essentially acting as a molecular fossil.
00:06:12.13 That is a compound that is diagnostic of an ancient cell,
00:06:16.24 and a particular cell type if it is to be useful to identify organisms or their metabolisms.
00:06:23.22 So the idea is that a long time ago
00:06:25.24 cells hanging around in the environment, be they in the water column or in sediments
00:06:30.13 eventually through their sedimentation, and then through various diagenetic processes
00:06:37.16 where these sediments were transformed and over time compacted and converted
00:06:42.08 into solid structures that we see today uplifted in mountains and in various areas around the world
00:06:49.27 contain yet remnants of the original molecules from the original cells that once upon a time inhabited these environments.
00:06:58.25 Now these structures are not identical, but they are very, very similar, and so
00:07:04.22 what we are looking at today is what we call the biomarker
00:07:07.10 and this is the molecular fossil of what we infer to have been the original compound.
00:07:12.20 So here you can see something that we call a sedimentary hopane that is the diagenetic...
00:07:18.12 And diagenesis is just simply the process of transforming a sediment over time into a rock.
00:07:24.25 This compound is the diagenetic consequence of transforming the parent compound,
00:07:33.25 which we call the hopanepolyol, which has this core pentacyclic ring
00:07:38.09 here with a tail that has a variety of moieties on various carbon molecules,
00:07:47.06 and this is one where we are just showing hydroxyl compounds at these last positions, but they come in a variety of forms.
00:07:53.20 The bottom line is that some of these chemical decorations are lost,
00:07:57.19 but the backbone carbon skeleton remains.
00:07:59.23 And because it is so structurally similar to the original compound
00:08:03.24 it is very reasonable to infer that we are looking at a fossil of this molecule.
00:08:08.16 Now here is just another way of showing that. This is essentially a cartoon of diagenesis.
00:08:15.25 Where through the water column these cells ultimately sediment down, get incorporated into sediments,
00:08:22.25 and these sediments through geological processes
00:08:25.19 over billions and millions of years of Earth history wind up transforming,
00:08:30.06 and these molecules within them are converted into their most basic form.
00:08:35.27 Now why do people pay attention to these particular molecules?
00:08:40.01 These 2-methylated bacterial hopanepolyols.
00:08:43.12 The reason is because of an important study that came out about a decade ago
00:08:47.20 where it was claimed that 2-methylhopanoids were biomarkers for cyanobacterial
00:08:53.00 oxygenic photosynthesis. And the rationale for this was that at the time this work was done
00:08:58.21 all of the surveys of microbial cells for this particular molecule,
00:09:04.00 and when I say particular I mean the pentacyclic part
00:09:09.15 of the molecule and a methyl group at the second position of the first ring.
00:09:15.21 The other aspects of the molecule are not as crucial to the diagnosis,
00:09:20.27 but this initial part, and particularly the methylation at this second carbon atom
00:09:26.17 really was crucial to the whole story that I am going to tell.
00:09:30.13 So back when this initial study was published the investigators
00:09:34.19 reasonably were able to conclude that these molecules might be biomarkers for cyanobacteria because of the
00:09:41.29 fact that they were unable to detect their production in organisms other than cyanobacteria.
00:09:47.12 This wasn't entirely true because there were a few other strains that even in this initial study were reported
00:09:52.24 to produce these molecules, yet it was argued that in their sedimentary context
00:09:58.03 the most likely progenitors of these molecuels would have been cyanobacteria
00:10:03.02 because they were shallow marine enviroments in which the fossilized compounds were found
00:10:07.27 that were consistent with the growth of a phototrophic organism in this type of a habitat.
00:10:13.19 And also, the argument that the concentration of these molecules in a type that was believed to be
00:10:20.06 the most preservable was by far the highest in modern day cyanobacteria
00:10:24.25 lending credence to the notion that these types of molecules, when seen in the rock record
00:10:29.06 very well may be reflecting an ancient cyanobacterial community.
00:10:33.15 However, whether or not this was an absolutely valid assumption was debatable for a variety of reasons.
00:10:42.13 One of these reasons is that not all cyanobacteria can make these compounds,
00:10:47.09 so they are certainly not essential for the ability of oxygenic photosynthesis.
00:10:52.06 And so the argument that just because cyanobacteria make them means that they have
00:10:56.13 anything to do with oxygenic photosynthesis
00:10:57.25 is a whole other story in and of itself and is something that really needed to be looked into more for validation.
00:11:06.24 The second reason that this needed to be looked into more is that cyanobacteria
00:11:10.19 are capable of other types of metabolisms beyond oxygenic photosynthesis.
00:11:16.22 Some of them are called facultative oxygenic photosynthesis organisms
00:11:21.14 in that they are capable of using other substrates beyond water
00:11:24.21 in photosynthetic processes such as sulfide, for example.
00:11:28.11 And in addition, they are capable of fermentation.
00:11:30.10 And so just because cyanobacteria make them, doesn't a priori mean that they have anything to do with photosynthesis.
00:11:36.03 Although they might. At this point in the story, nobody knew, and so it merited looking into.
00:11:40.20 And so we got interested in this, and we thought that we would begin our studies
00:11:44.11 by coming up with just some rational criteria for what would constitute a robust biomarker.
00:11:48.29 The first of course is one I am not going to touch on because this really belongs to the province of geologists.
00:11:54.13 And that is that the biomarker must be indigenous to the rocks that it is meant to represent.
00:11:59.26 There's actually great debate about this point, but let's just assume for the sake of argument that when we find these fossils
00:12:06.11 in ancient rocks that they really are as old as we think they are.
00:12:10.15 And we will let the geologists work out indeed whether that's true,
00:12:13.13 but I think that it is fair enough to say that many of these samples
00:12:17.06 now there isn't much of a question. These molecules really are for instance 2.4-2.7 billion years old.
00:12:26.17 So let's just grant that.
00:12:27.14 The second part which now becomes of relevance for biologists is that they must have a unique distribution
00:12:34.05 amongst modern organisms, or they must have a clear evolutionary history
00:12:38.18 where it is obvious from whence they originated.
00:12:41.21 What were the first type of organisms that were able to produce them and/or the third point,
00:12:48.16 which I think is actually the most crucial point,
00:12:51.10 that they have a specific and conserved biological function that's related to the process of interest.
00:12:57.16 And in our case for today's lecture, we are talking about oxygenic photosynthesis.
00:13:01.22 So let's begin now with this second point.
00:13:05.28 Do these molecules, these 2-methylhopanoids have a unique distribution?
00:13:09.19 So to begin to look into this we decided to go to the environment
00:13:14.08 and collect some diverse types of phototrophic organisms to work with.
00:13:17.25 And we began by working with various cyanobacteria
00:13:21.04 that we surveyed for their ability to produce these 2-methyl compounds.
00:13:25.06 As I said, not all of them do, so we picked ones that were abundant producers as our reference strains,
00:13:31.05 and then because we had also been doing other studies in my lab
00:13:34.16 looking at the ability of anoxygenic phototrophs to catalyze the oxidation of iron,
00:13:42.08 under anoxygenic conditions, we decided just for fun we would begin to test
00:13:48.06 whether or not they could produce these compounds as well,
00:13:50.12 thinking that this would be a negative control, because previous reports had shown that related strains were,
00:13:57.17 under the conditions these previous investigators used, not able to generate these compounds.
00:14:03.18 And so here is where we had one of the surprises that occur once in a while in science
00:14:08.19 that are very exciting and at first cause you great distress
00:14:11.12 because you are not sure whether or not it's an artifact.
00:14:14.14 And so this happened when my student Sky Rashby made the surprising discovery
00:14:18.11 that one of our "negative control strains", one that we called Rhodopseudomonas palustris
00:14:23.03 was capable of making these 2-methyl hopanepolyols, that's what this acronym here means,
00:14:28.17 2-MeBHP stands for 2-methylated bacterial hopanepolyol.
00:14:33.06 In concentrations as great as its cyanobacterial counterparts.
00:14:38.02 Now you can be assured that we did all of our proper controls to make sure we weren't contaminating the cultures,
00:14:43.25 and this was a very reproducible, very robust result.
00:14:48.03 And what was really quite significant is that this molecule here,
00:14:52.15 and here you can see liquid chromatographic spectrum, and now a mass spectrum
00:14:58.14 of these peaks, was identical between our cyanobacteria positive controls
00:15:04.07 and what we thought was going to be our negative control.
00:15:06.20 What was really crucial here is that the production of these molecules by our anoxygenic phototroph
00:15:13.24 was conditional upon the manner in which we grew them.
00:15:17.12 And I think this is a very important point that is worth emphasizing.
00:15:22.21 And that is, oftentimes in these interdisciplinary fields
00:15:26.07 they begin with a group of investigators who are excellent at a particular type of measurement
00:15:31.12 but not necessarily excellent at all, because who is? You know we are all specialists. We are all good at doing what we do.
00:15:39.20 And so the pioneering scientists who began this work were outstanding organic geochemists,
00:15:45.06 but were not people who routinely grew microorganisms in their own laboratories
00:15:49.00 so they were dependent upon getting strains from their colleagues
00:15:52.27 who would send them microbial samples grown up in whatever random growth condition
00:15:57.04 they happened to be doing at the time that they shipped the strain.
00:16:00.00 However, today, when we grow them in our laboratory, being microbiologists,
00:16:04.17 we are aware of the metabolic versatility of these organisms and are able to grow them under a variety of conditions.
00:16:10.04 And because of this we were able to find that it was under certain conditions
00:16:15.05 that these organisms made the molecule, and had we only looked at them in a more narrow window,
00:16:21.09 we would have missed their production entirely.
00:16:23.09 So there is an important lesson here, and this nice cartoon
00:16:26.15 of this filamentous cyanobacterium that is supposed to represent the cyanobacterium Nostoc punctiforme
00:16:32.17 that I'll show you later in the talk
00:16:34.06 is saying, "Do you make 2-methylhopanoids?" to our favorite model anoxygenic phototroph,
00:16:39.19 Rhodopseudomonas palustris, and she is answering, "Yes, I do."
00:16:42.10 This very nice cartoon was made by Sky, who is a very talented artist.
00:16:46.16 Now an actual image of this purple bacterium is shown here
00:16:51.25 in a transmission electron micrograph, taken by my postdoc Ryan Hunter,
00:16:55.18 that he overlaid upon sediments from the Sippewissett Salt Marsh in Woods Hole, Massachusetts.
00:17:01.21 And the reason we did this is because as I've been saying,
00:17:05.00 the geological record ultimately is emanating from sediments such as these,
00:17:10.03 where today we find purple bacteria and other types of phototrophs growing.
00:17:13.23 And one can infer that these types of environments also were good homes
00:17:19.11 for these organisms back billions of years ago.
00:17:22.05 And so what we are looking at when we are looking at the fossils
00:17:25.11 are ancient remnants of these types of habitats.
00:17:28.06 So what we thought we would begin to do would be to study Rhodopseudomonas palustris
00:17:33.01 to ask whether or not we could gain insight into the molecular biological basis for the production of these compounds,
00:17:40.21 and have this help us parse whether or not they were accurate biomarkers
00:17:44.09 for cyanobacteria or, and/or, oxygenic photosynthesis.
00:17:49.00 Now R. palustris is a terrific organism to use for many reasons. It grows rapidly.
00:17:55.02 It is metabolically versatile. It's genome is sequenced.
00:17:57.17 And it is amenable to genetic manipulation. And so it's a system primed for discovery
00:18:03.19 in terms of the biosynthesis and the cellular function of 2-methyl BHPs.
00:18:08.01 So one of the first things that we wanted to answer was whether or not
00:18:11.14 there was a specific enzyme responsible for methylating hopanoids
00:18:15.05 at the C2 position because given the fact that these molecules were produced very conditionally
00:18:22.14 upon specific quirks of how one were to grow them, what would be desirable
00:18:29.08 in terms of asking the question which organisms writ large are able to make these molecules
00:18:33.25 would be to bypass this growth requirement and simply be able to go to an organism's genome
00:18:39.00 to see if there were some gene that were diagnostic, that could predict the ability to make these molecules.
00:18:44.17 So Paula Welander, a postdoc in my laboratory,
00:18:47.07 sat down with the genome of Rhodopseudomonas palustris
00:18:50.01 and through some very clever bioinformatic work identified a whole part of the chromosome that encoded genes
00:18:57.03 that she suspected might have something to do with hopanoid biosynthesis.
00:19:00.13 And that's because in the middle of this cluster there is a gene annotated as shc that had previously been shown
00:19:07.19 to encode an enzyme responsible for the first step in hopanoid biosynthesis,
00:19:13.11 the cyclization of squalene into the first pentacyclic ring.
00:19:20.16 Now what we wanted was the enzyme responsible for a later part in the biosynthetic pathway
00:19:28.27 and that is the methylation at C2 because a wide variety of bacteria are capable of making hopanoids in general,
00:19:35.03 but what had been thought, as I told you, to be the diagnostic feature was this methyl group at the second position.
00:19:42.03 So if we could find a methylase that was specifically responsible for putting this methyl group on that position
00:19:49.02 we might be in a position to ask whether or not different organisms beyond cyanobacteria and this one freak
00:19:56.08 discovery we made, were capable of also making 2-methylhopanoids.
00:19:59.12 So to do that Paula looked at this genomic region, and she identified a gene over here
00:20:07.26 that seemed to have attributes that might be consistent with its product being
00:20:13.17 capable of catalyzing this methylation reaction.
00:20:16.21 And so to test that she made a clean in frame deletion of this gene,
00:20:22.10 and then she asked whether or not the mutant
00:20:24.19 strain that she called delta 4269 was capable of making methylated hopanoids in comparison to the wildtype.
00:20:32.22 And so here what you are looking at is a liquid chromatogram showing peaks of elution
00:20:38.03 of various types of hopanoid structures.
00:20:40.19 These over here are called...this one is diploptene, and this one
00:20:45.19 has a methyl group at the 2 position, so it is called 2-methyldiploptene
00:20:49.14 and this is a different type of hopanoid, with a slightly different R group on the end.
00:20:57.07 And again here, this peak that elutes first is the 2-methylated version of the molecule.
00:21:02.06 And as you can see when you compare it to the mutant that Paula made,
00:21:05.15 there is no peak here in the foreground showing that the 2-methylated versions
00:21:11.09 of these hopanoids are not being made in this mutant background.
00:21:14.04 Now when she complements and adds back to this mutant strain the wildtype
00:21:18.21 gene, then again she sees, indeed, just where she should, the peaks
00:21:23.29 that indicate the production of the methylated versions of those hopanoids.
00:21:27.22 So this was a very nice demonstration that indeed this methylase was responsible for
00:21:32.24 producing these methylated hopanoids.
00:21:36.02 Now in collaboration with another postdoc in the lab, Maureen Coleman,
00:21:40.27 Paula and Maureen went on to show that this methylase gene that they dubbed hpnP,
00:21:46.22 was much more broadly distributed than we had suspected.
00:21:50.28 And so what matters here is not the individual names of these organisms,
00:21:55.29 by any means, but simply the fact that what you are looking at is a complicated tree
00:22:02.04 that's drawn using the 16S ribosomal DNA sequence for a whole variety of organisms.
00:22:09.04 And what you are looking at now is in blue those organisms on this tree that are capable of producing hopanoids in general.
00:22:18.06 And there are many, as I said.
00:22:20.06 But the really key piece is that here, which is this outer ring
00:22:24.12 in which you can see in three different groups, in the cyanobacteria, in the acidobacteria,
00:22:32.17 and down here in the proteobacteria, evidence for the hpnP gene from the genome.
00:22:39.20 And this evidence is based on a very stringent cutoff where we are asking for a very high degree of similarity
00:22:47.05 in sequence identity between putative hpnP sequences
00:22:52.03 and our sequence of hpnP that we have shown is required for the methylation at the C2 position.
00:22:59.11 Now the other thing to note is that in every case that these sequences have been found in the genome
00:23:11.06 where the measurements of the actual ability to make the compound have been made, we have found
00:23:16.18 a 100 percent correspondence. And inversely, we have never found an instance
00:23:23.05 for an organism that is capable of making 2-methylated BHPs where they don't contain this gene.
00:23:29.03 So while of course it is possible that in the future another type of methylase will be discovered,
00:23:35.17 right now there is a 100% correspondence between position of this gene in the genome
00:23:40.26 and the ability to make the compound as measured directly.
00:23:44.28 So it appears as though it is a fairly robust indicator.
00:23:48.03 All right so, there are a few things to note here, as I said before, not all cyanobacteria make this.
00:23:54.06 So a priori, it is certainly not required for oxygenic photosynthesis.
00:23:57.00 The question is whether...and number two, more than cyanobacteria make it. It is not just a case of one random organism
00:24:05.02 being a flier. No. We find that there are many other types of bacteria here in this proteome,
00:24:12.02 alpha-proteobacteria family, that are capable of producing these compounds.
00:24:16.00 Some of these actually have even been shown in that very original paper to produce the methylated hopanoids.
00:24:22.04 It just had been claimed that they weren't producing them in abundance that was relevant for diagenesis.
00:24:27.03 And so that deserves a bit of re-examination.
00:24:29.27 But here is this whole other group, the acidobacteria, that hadn't previously been appreciated to make these molecules.
00:24:38.25 And I should say that this is just the tip of the iceberg, because these are only the genomes
00:24:42.08 of microbes that currently have been sequenced, which represent a minute fraction of the microbial diversity
00:24:48.01 in nature, and so now we have the tools where we can go out into the environment and ask more broadly,
00:24:54.01 where do we see these sequences? What type of organisms are likely containing them?
00:24:58.22 And we'll get a much better picture of the phylogenetic diversity of the organisms that make them.
00:25:03.00 However, to get back to the central point in terms of whether or not this is a robust biomarker,
00:25:09.26 even though today we see that these molecules are distributed amongst many organisms,
00:25:15.14 they still might be a valid biomarker of cyanobacteria
00:25:17.28 provided we can have some good evidence suggesting
00:25:23.05 that their root evolutionarily was within the cyanobacterial group.
00:25:28.11 So to get at that question of what is the root of this enzyme,
00:25:32.07 where was it first invented, Maureen Coleman decided to look at the phylogenetic history of the enzyme family.
00:25:39.08 So she made a tree where she was able to show, and now this is an unrooted tree,
00:25:44.03 the different branches of the organisms that we know can make this.
00:25:48.27 All right, so these are two different groups of proteobacteria. Here we have the cyanobacteria.
00:25:52.20 And here we have the acidobacteria.
00:25:55.07 And what we wanted to know was which of these groups was the first to invent this enzyme.
00:26:01.01 Now here is where it became complicated, and also where it became very important to do a rigorous
00:26:06.21 statistical analysis to align these sequences in various ways, use different algorithms,
00:26:12.18 to infer the most parsimonious evolutionary path to their divergence.
00:26:19.01 And when Maureen did this, quite literally by doing an in silico experiment, by varying a variety of parameters,
00:26:26.24 none of which was clear what was the absolute correct assumption to make,
00:26:32.00 but all of which would affect the topology of the trees that ultimately would result.
00:26:36.29 What she found was that the root, at least at present, is ambiguous,
00:26:41.19 that statistically there was as good support for these three different topologies,
00:26:47.14 that led to very different answers.
00:26:49.26 The first where the ancestral root of this methylase would be in the cyanobacteria.
00:26:54.01 The second where it was quite simply unresolved,
00:26:58.08 where there was no clear branching pattern,
00:27:00.27 of which subset would have been the earliest one to make this enzyme,
00:27:06.11 or the opposite result where the ancestor would be inferred to be an alpha-proteobacterium.
00:27:12.18 We hope that in the future as more sequences are gained we will be able to,
00:27:17.21 with greater confidence, resolve between these three options, but what we can confidently say
00:27:21.28 now is that we cannot with confidence interpret that cyanobacteria were even the evolutionary inventors of this.
00:27:30.11 And so the case remains open whether or not in the future they will be proven to be so,
00:27:35.25 but at present it is invalid to argue that there is an absolute certainty
00:27:41.12 that these molecules are biomarkers even for the cyanobacteria themselves,
00:27:46.03 saying nothing about oxygenic photosynthesis.
00:27:48.05 So let's cut to the chase and talk about oxygenic photosynthesis
00:27:51.02 because that is really what motivated this all along.
00:27:54.18 Could we find a biomarker that would give us insight into the process of oxygenic photosynthesis?
00:27:59.15 And of course, just because an organism makes a molecule, doesn't necessarily
00:28:02.28 mean that it has anything to do with a particular function.
00:28:06.04 And so what we wanted to ask was whether or not there was a connection between
00:28:10.22 the ability to produce this molecule and photosynthesis directly.
00:28:15.05 So, as I told you, hopanoids resemble sterols.
00:28:20.16 And if you take a look at the biosynthetic pathway
00:28:25.00 for cholesterol over here and compare it to bacteria hopanoids over here,
00:28:32.13 you see that they go through very similar structures.
00:28:36.22 Of course the rings are different between them, but they are reminiscent.
00:28:42.15 And so it would be reasonable when thinking about the universal functions for these molecules
00:28:48.01 that we would turn to sterols for some inspiration.
00:28:50.26 And this is a very fascinating field in cell biology in eukaryotes,
00:28:56.06 and I am sure there are some interesting commentaries on this in other iBioSeminar series that you can refer to.
00:29:01.13 Very briefly what I will just say now is that it has been demonstrated
00:29:05.10 that sterols in eukaryotic membranes have roles in both membrane fluidity and integrity.
00:29:10.12 They can play roles in membrane curvature. They are also important
00:29:16.05 in creating what are called lipid rafts, that involve the recruitment
00:29:20.13 of specific lipids and proteins to particular places within the membrane.
00:29:24.06 All of these are very active areas of research in eukaryotic cell biology,
00:29:28.01 and one can reasonably ask whether or not in bacterial and in archeal,
00:29:33.13 or even microbial- eukaryo systems these hopanoid molecules
00:29:39.02 that resemble sterols might play similar functions.
00:29:41.26 And one can go further to ask whether or not in any way these functions are related specifically to oxygenic photosynthesis.
00:29:50.01 So, one thing that we can do to begin to address this is simply to figure out in different types of microbial cells
00:29:58.16 where hopanoids reside. And the point here is that it is not obvious,
00:30:05.05 because microbial membranes are actually very complex,
00:30:08.07 particularly in phototrophs where we have these
00:30:10.17 beautiful inner cytoplasmic membrane lamellar invaginations.
00:30:15.11 We can have other types of invaginations
00:30:17.06 that are either vesicular or tubular, there are many different types of
00:30:20.20 internal membrane structures that can form,
00:30:22.22 and as I said this is a wonderful opportunity for future research in microbial cell biology.
00:30:26.28 But getting back to the focus here, the question is, and in this cell,
00:30:31.29 what I am showing you is a thin section of my favorite organism Rhodopseudomonas palustris,
00:30:36.06 where do the hopanoids reside? Are they in the outer membrane or are they in the cytoplasmic membrane?
00:30:40.27 Are they in this membrane called the inner cytoplasmic membrane,
00:30:46.14 which is where the photosynthetic machinery is housed?
00:30:48.27 And the same question can be asked of cyanobacteria, where you see these inner cytoplasmic membranes,
00:30:55.04 which also host the photosynthetic machinery in cyanobacteria.
00:30:59.20 And this is an organism that isn't even photosynthetic,
00:31:02.17 but does indeed produce 2-methylated hopanoids
00:31:06.13 and as you can see, it has complex membrane systems as well.
00:31:09.10 So where are the hopanoids, in which of these membrane systems?
00:31:12.03 And just for the remainder of this time, I am only going to focus on showing you work
00:31:16.07 we've done using a representative cyanobacterium called Nostoc punctiforme.
00:31:20.28 So Nostoc punctiforme is a terrific microbial system for studying cellular differentiation.
00:31:26.24 And the reason for this is that there are three states it can exist in, as the vegetative growing cells,
00:31:32.16 some of which can differentiate to form specialize structures called heterocysts,
00:31:36.27 and this is where nitrogen fixation occurs in this organism.
00:31:39.09 But these organisms are also capable of creating a spore like state,
00:31:45.10 that is known as an akinete, and this is a very stress resistant cell type
00:31:49.26 that forms under stressful conditions that is not metabolically active. It is metabolically quiescent.
00:31:55.13 So Dave Doughty, a postdoc in my laboratory decided to take Nostoc
00:32:01.11 as a beginning entry way into understanding
00:32:04.25 where hopanoids would partition in cyanobacteria.
00:32:07.28 And what he decided to focus on were the different membranes that I just described:
00:32:12.14 the outer membrane, the inner membrane, and the inner cytoplasmic membrane
00:32:17.15 in the vegetative, heterocyst, and the akinete cell types.
00:32:21.05 And what you can see when you do these thin sections of the organisms
00:32:24.16 and look at them under electron microscopy,
00:32:26.24 is that the cell types change quite dramatically.
00:32:30.02 For example, there are very few, what he is labeling here as thylakoids,
00:32:34.14 this is just borrowing a term from eukaryotic cell biology
00:32:37.20 but what this is also really talking about here is the inner cytoplasmic membrane
00:32:42.15 that these cyanobacteria have that houses the photosynthetic apparati.
00:32:47.28 They have very few when they are in the akinete state, which makes sense, because these are resting cell types.
00:32:53.04 The heterocysts have a much thickened cell envelope, and these are the cells in which nitrogen fixation occurs.
00:33:01.15 And this is just a normal view of a vegetative cell,
00:33:04.12 where you have what Dave calls a thylakoid membrane, and what I have been calling the inner cytoplasmic membrane.
00:33:10.04 As well as an outer membrane and an inner membrane in the overall cell envelope.
00:33:15.27 Now what we wanted to ask was depending upon the way in which the cell was grown,
00:33:22.10 would we find that hopanoid content and also hopanoid localization to these three membranes change?
00:33:28.13 And the answer was dramatically yes.
00:33:31.04 And so what Dave showed was when he was able to grow them as vegetative cells,
00:33:36.14 as heterocysts. Isolate the heterocysts, and then grow them so that they went into the akinete cell type
00:33:43.24 and then fractionate each of those three different cell types with respect to the cytoplasmic membrane,
00:33:48.06 the thylakoid membrane, and outer membrane,
00:33:51.20 that for these cell types the concentration of hopanoids, micrograms of hopanoids per milligram of total lipid,
00:33:58.20 varied quite dramatically.
00:34:01.01 And what I'll have you note here is that for the cytoplasmic membrane and the thylakoid membrane
00:34:05.08 the scale is much smaller than for the outer membrane.
00:34:09.24 Here this is only going up to ten, and here it goes up to sixty.
00:34:14.11 And so while indeed we do find some of these hopanoids in akinetes
00:34:20.08 and in vegetative cells being generated in the thylakoid membrane
00:34:25.15 by far the greatest concentrations are in the outer membrane of the akinetes, which are the resting cell types.
00:34:33.15 So as I told you, akinetes can be very resistant. They are survival structures.
00:34:40.08 But what I want to add to that description is the fact that
00:34:43.06 they can develop when cells are grown completely in the absence of light.
00:34:48.14 And so while in this experiment Dave did he'd originally grown them up photosynthetically,
00:34:53.02 and then subjected them to starvation conditions so that they went into the akinete type.
00:34:57.04 They do not need to ever go through the photosynthetic phase in order to become akinetes.
00:35:02.20 Therefore, since the majority of the methylated hopanoids in this organism
00:35:08.04 are localized to the outer membrane of the akinetes, one can infer that
00:35:13.19 the most likely source of 2-methylated hopanoid deposition into sediments, at least from this organism,
00:35:19.17 would be from cells where photosynthesis is not occurring.
00:35:24.11 And because of this we can go back and interpret the rock record and ask the question,
00:35:32.06 when we find both these molecular fossils of 2-methylated hopanoids
00:35:36.17 as well as these morphological fossils that paleontologists who are very clever and able to look at
00:35:44.04 these images and actually tell you when it is an akinete versus some other type of cell
00:35:49.03 and date them to very old times in the rock record.
00:35:54.00 Here we are talking about billions of years, so 1.5, 1.6, and 2.1 billion year old samples
00:36:00.12 where the akinete cell types as diagnosed by these paleontologists
00:36:05.13 are known to have been produced, we can ask whether or not
00:36:11.04 the 2-methylated hopanoid record that we find at similar ages
00:36:16.11 might have been produced through the shedding of the akinete envelope
00:36:19.12 into the sediments and may be reflecting a stress response
00:36:22.23 as opposed to anything to do with oxygenic photosynthesis per se.
00:36:26.27 That said, there is still more work to be done in examining whether there is some type of indirect connection
00:36:34.26 with oxygenic photosynthesis. There may be.
00:36:37.13 And we have a lot more to discover, so this is a very exciting field to be in
00:36:40.22 and will remain a very fruitful area of research for many years.
00:36:44.15 So to summarize what I hope I have been able to show you through an example
00:36:49.00 is how we can re-examine molecules that have been used as biomarkers for ancient metabolisms,
00:36:55.16 and ascertain whether or not these are valid biomarkers.
00:36:58.24 In this particular case, we have shown that these compounds
00:37:01.23 2-methylated bacterial hopanepolyols, or hopanes, as they are called in the rock record,
00:37:07.00 are likely to have been generated by many diverse bacteria.
00:37:11.02 And we can not yet say which type of organism invented the ability to make them.
00:37:15.19 So the jury is still out whether they can be used as biomarkers of cyanobacteria.
00:37:19.01 That said, even if cyanobacteria did invent them first,
00:37:24.29 it is not at all clear that they have anything to do with oxygenic photosynthesis
00:37:28.15 and in fact, all of the available evidence really would most reasonably lead you to conclude otherwise.
00:37:33.20 And that is because cellular localization studies show that they are mainly in membranes
00:37:40.28 that do not have anything to do with active cells,
00:37:43.27 nor do they necessarily have to reflect going through a photosynthetic process.
00:37:48.28 And in experiments which I don't have time to show you here,
00:37:51.21 we've been able in Rhodopseudomonas palustris to actually knockout these methylated compounds,
00:37:57.13 and find that even for the process of anoxygenic photosynthesis
00:38:01.04 they are not required and so, it doesn't appear that they play a direct role in metabolism.
00:38:07.08 But what it does seem to be important is that they are involved in stress response,
00:38:11.06 and likely this is manifesting through effects in membrane permeability and membrane fluidity.
00:38:17.18 Potentially there are other roles, and we await future discoveries to eliminate the full menu of options
00:38:25.06 that these molecules are serving in terms of their biological function in modern cells.
00:38:29.26 All right, so to summarize what I hope I have been able to show you from this story
00:38:33.23 is that we can use studies of modern bacteria to gain insight into the genesis of ancient metabolisms.
00:38:40.06 And while there is still a lot more work to do, we have a very exciting
00:38:44.25 future ahead of us where we have these remarkably geostable
00:38:48.14 compounds that hang around in rocks that are incredibly old, going back over two billion years,
00:38:53.21 and we need to be able to interpret them rigorously. And so in the case of these 2-methylated bacterial hopanes,
00:39:01.09 it appears that they really are no longer the best indicators of oxygenic photosynthesis,
00:39:06.10 however, they might be good indicators of microbial stress responses and other things.
00:39:12.23 And so in the future we hope to understand what their biological functions
00:39:17.15 are in both cyanobacteria and the other organisms that make them,
00:39:22.07 and be able to utilize these molecules as biosignatures of wonderful inventions in the history of cell biology.
00:39:28.23 So with this, I would like to acknowledge the people in my lab
00:39:31.29 that I have been referring to who have done this work.
00:39:34.08 It's been a collaboration over many years, and it began at Caltech with the student Sky Rashby,
00:39:40.05 co-advised by my colleague Alex Sessions, and in collaboration with my colleague Roger Summons at MIT.
00:39:44.18 And then continued by two postdocs, primarily Paula Welander and Dave Doughty.
00:39:52.04 Paula doing the work in Rhodopseudomonas palustris, and Dave with Nostoc,
00:39:56.08 and now continuing with Rhodopseudomonas, helped out by phylogenetic work with Maureen Coleman
00:40:02.02 and microscopy by Ryan Hunter.
00:40:03.26 And the Howard Hughes Medical Institute, NASA, and the NSF have contributed to supporting our research.