Microbes are diverse, ancient, numerous and ubiquitous. In part 1, Newman gives an overview of these four key points. She presents mind-boggling data on the numbers of microbes inhabiting the earth, as well as the environments in which they can survive, and indeed, thrive. Both fossilized and modern microbes come in fantastically diverse physical forms and this diversity extends to their metabolism. Newman explains how geobiologists can deduce information about ancient microbial life by studying rocks formed on earth billions of years ago.
In the last part of her lecture, Newman describes efforts to date the evolution of oxygenic photosynthesis (photosynthesis that uses water as a substrate and produces oxygen as a product) in the ancient rock record using a particular type of a molecular fossil, or “biomarker”. She explains the importance of identifying a robust biomarker, and describes various criteria that should be applied to this pursuit.
Microbial Diversity and Evolution
Concepts: Microbial diversity, fossilized and modern microbes, metabolism evolution in microbes, rRNA phylogenetic tree of bacteria
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.
- Compare and contrast the insights into ancient microbial metabolism given by morphological, molecular, or genomic biosignatures. Is one the best?
- This lecture presents many examples of communities of microorganisms living within more advanced multicellular organisms. Why do you think that this is such a common theme in nature?
- How might our growing knowledge of the human ‘microbiome’ (there are actually more bacteria cells than human cells in our body) change how we treat disease?
- After hearing about the diverse and ubiquitous nature of microbial life on Earth, are you more or less confident that extraterrestrial life exists?
Interpreting Molecular Fossils of Oxygenic Photosynthesis
Concepts: Evolution of oxygenic photosynthesis
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.
- Is there a direct connection between the ability to produce 2-Me hopanoids and oxygenic photosynthesis?
- How does the story about R. palustris emphasize the importance of running experiments under varied conditions?
- If 2-Me hopanoids are not unique to cyanobacteria, then why does Dr. Newman suggest that 2-Me hopanoids could still be a biomarker of the origin of cyanobacterial photosynthesis?
- Why is it of interest to identify a specific role for 2Me-hopanoids in living cells?
Discussion Questions for the Paper
- How might the 2-methylation of hopanoids permit adventitious adaptation?
- Why are the findings of previous hopanoid studies not likely to reflect the true roles of hopanoids in vivo?
- What evidence suggests that methylation at the C-2 position of hopanoids promotes fitness under environmental stress?
- What are the challenges to interpreting the meaning of any ancient molecular fossil?
Dr. Newman is a Professor in the Divisions of Biology and Geological and Planetary Sciences at the California Institute of Technology. When Newman began her undergraduate studies at Stanford University she wasn’t sure she was going to be a scientist because she was interested in a variety of different fields. In fact, she received her… Continue Reading
is this the evolution and diversity of microbial life?