Dianne Newman (CalTech) Part 1: An Overview of Microbial Diversity and Evolution

Hello, and welcome to iBioSeminars. My name is Dianne Newman, and I'm a professor in the Divisions of Biology and Geology and Planetary Sciences at the California Institute of Technology, and I am also an investigator at the Howard Hughes Medical Institute. So I am going to be giving you a lecture in three parts today, and this is part one, which will be a very general overview on microbial diversity and evolution. In part two, I'll tell a specific story about a modern example of a microbial metabolism that's quite interesting and very important in affecting the geochemistry of the environment with regard to arsenic geochemistry. And in part three, I'll talk about work we've been doing that is more directed at understanding a metabolism that evolved in the past, namely oxygenic photosynthesis. But let's start with an overview now and consider four important points about microorganisms and their history.

And I am going to walk you through each of these four points. So the microbial world is really quite remarkable and my goal in this first overview is to leave you with an impression of its diversity, its antiquity, and how abundant and ubiquitous this world is. So let's begin with antiquity. When we think about the evolution of life, oftentimes we think in terms of macroscopic fossils such as the ones that you see here. And it is pretty clear when you look at these rocks, that something living was present on Earth when they formed. In this panel over here you see a fossil of some type of algae. It is not clear exactly what type, but it is inferred to be an alga. And this shape here is known as a trilobite. And this section of rock that you are looking at is one of the most famous fossils on the plane. It is called the Burgess Shale.

It's found in Canada, and it dates to what we call the Cambrian explosion, which occurred roughly half a billion years ago around five hundred and sixty million years ago. So we can certainly claim when we look at rocks of this age that life was present. But if we want to think about the evolutionary history of life, over a much larger time span of billions of years, given that the Earth is 4.6 billion years old, we need to step back in time and look at more ancient rocks. And when we do this, the shapes suddenly change, and it becomes not quite as evident that we are looking actually at fossilized versions of life, and yet we are. So for instance, take this rock as an example. Here you see these dome-like structures, and these are vestiges of a type of microbial community forming in a shallow marine environment that became lithified and left these domal structures, and we call these structures stromatolites.

Now this particular rock that you are looking at is about 3 billion years old and is from South Africa. But these rocks can be found all over the world, and they occur throughout Earth's history, going back as far as 3.4 billion years. However, when we go even further back in time, for example, back to 3.8 billion years, you can see ore deposits that one might not intuit immediately had anything to do with microorganisms, and yet they do. They indeed record a history of microbial activities that was quite profound, so profound that it quite literally transformed the planet. And this is one beautiful example. So what you are looking at here is actually a 2.4 billion year old quarry. This is in Western Australia in the Hamersley formation, and this is known as a banded iron formation. And they're extremely important today because they constitute the world's largest source of iron ore. But they also record a remarkable history of the evolution of metabolism. Now how can this be? How do these massive rock quarries tell us anything about microbial life? Well, when you think about what they actually constitute they are made up of iron minerals, as well as other minerals cherts, which is a type of silicon oxide, intermixed with these iron species, but for now let's just focus on the iron.

So how did this iron get into this big deposit that you see here? Well, it began a long time ago in ancient seas, in the form of ferrous iron that's called Fe2+. And then some process, which I'll get to in just a minute, oxidized this ferrous iron to ferric iron, and at that point it could react with constituents in the waters such as hydroxyl species, to form iron minerals, such as this one: ferric oxyhydroxide, rust. And over time this mineral transformed and changed into different types of minerals, became compacted, and mingled with others and wound up in these rocks that we today know as banded iron formations. But this initial step here is the critical one in terms of giving us some insight into microbial activities on the ancient Earth. And let's think about two scenarios where microorganisms might have been involved.

The first scenario is one where a very primitive type of photosynthetic organism, well, I should say primitive in quotes, because actually this metabolism is remarkably sophisticated. Nonetheless, this is primitive in the sense that it is a type of photosynthesis that does not generate oxygen. Rather it is called anoxygenic, meaning that there is an electron donor, in this case ferrous iron, that is oxidized to ferric iron and that powers the reduction of inorganic carbon, CO2, to biomass. And you can see this is a very dramatic metabolism when it occurs because all you need is light, microorganisms, and ferrous iron, and a few other things to help them get going, but those are really the three most important ones in a bottle here, with, as I said, a few nutrients added so they can do their thing, and when light is shined on this bottle, these organisms very rapidly are able to oxidize the iron.

And they produce rust, and you can see the rusty color here in this bottle. And this rust is exactly the type of iron that is the predecessor of the minerals that constitute these banded iron formations. Now in the middle you see these organism growing on a different electron donor, and I'll get to what I mean by an electron donor and an electron acceptor later in this lecture.

And in this case they are utilizing hydrogen as an electron donor, and the pink color you see is due to photosynthetic pigments in their membranes that enable them to harvest light and grow in this way. So this scenario, as I said, is one that is catalyzed by organisms that do not generate oxygen. They are anoxygenic phototrophs capable of oxidizing iron in a photosynthetically mediated process under environments where no oxygen is present whatsoever, and yet these ferric minerals can form. Now scenario two, that is entirely different is one where the organisms that ultimately catalyze the precipitation of these minerals were producers of molecular oxygen, and these are the cyanobacteria that you can see here that were critically important in the history of the evolution of metabolism and quite frankly also in changing the overall chemistry of the Earth including its atmosphere because they evolved the ability, the remarkable ability, to use water as an electron donor in photosynthesis, oxidize it to molecular oxygen, and through this process, power the reduction of CO2 to biomass.

Now once they produce this oxygen, the oxygen chemically would have been able to react with ferrous iron, oxidizing it to ferric iron, and then this in turn would go down the pathway to precipitate these rusty minerals I showed you. So here we have two options: one scenario where no oxygen is involved, and a second scenario where oxygen is mandatory. And both of these are biological processes. So how do we distinguish between them if we are interested in understanding the types of organisms that were present on Earth in the remote past? Well this is quite a challenge, indeed, and there will be many years of investigations in the future in order to really pin this down. And it is a great field to get into if you are a beginning student and interested in both biochemistry and evolution, but what I'll say just for now is that we know from a variety of indicators that somewhere between 2 billion and 3 billion years old it is very probable, indeed it is almost certain, that the process of oxygenic photosynthesis arose.

But when exactly this happened and how the evolutionary events came together such that these anoxygenic phototrophs that can utilized reduced substrates such as hydrogen, or sulfur species, or iron as electron donors in photosynthesis morphed into a more sophisticated type of phototroph, that was capable of using water as an electron donor, the cyanobacteria, which in turn, are what became the plastids, the chloroplasts that we find in modern marine algae and also of course, in plants that are very well known for their ability to do oxygenic photosynthesis. We do not know. We do not know when this happened. And in my third lecture in this series, I will discuss ways that we can begin to approach this problem. But it's a profound question, and what I would like to leave you with now is just the simple message that these very ancient rocks, such as these banded iron formations, here are holding clues to a mystery that we have to unravel. And it is through tools of modern biology that ultimately we hope to get there.

All right, now as I said the history of microbial life extends very far back in time, as far as 3.8 billion years as we currently estimate, but this might have been even earlier for all we know. How do we decipher when particular microbial metabolisms evolved and what types they were? Well, this indeed is extremely challenging. And there are three primary ways that we can gain insight into the microbiology of the past through using either morphological, molecular, or genomic, which is of course a form of molecular biosignatures.

And these are very different in what they can tell us. So the first two, morphological and molecular, are important because they can be concretely linked to rocks, old rocks, that we can date. And because of this when we see a particular form, this is being held in the hand a sample of stromatolite. This is at a very different scale here. You are looking at a thin section of a rock, and that is true for these images below where the scale is about 1 millimeter, in this image, and it is even smaller down here.

The structures that you observe have been interpreted as being vestiges of ancient life for various reasons. But this interpretation is often ambiguous, and it is a challenge to be able to come up with unambiguous biosignatures simply on the basis of their shape. And so geobiologists, those interested in seeking to understand life in ancient times, have turned recently to what we call molecular biosignatures that come in two forms: either organic biosignatures, or some type of inorganic biosignature, often expressed as a ratio of different isotopes in a sample. Now this in turn is challenging as well, but it may be the best way that we can gain more specific insight into different types of metabolisms, by looking at actually the chemistry what is left in the rock and being able to deduce through finer scale analyses whether or not this chemistry was one that was uniquely imparted by a biological process. Lastly we can think of genes as fossils, and the genomic record has been crucial in establishing the diversity of life on the planet, as I'll get to in a little while in this lecture, but it also helps us understand the relatedness of different enzymatic functions and how they evolved from one another.

While this does not give us a concrete date when these metabolisms evolved, it does provide us with an ability to look at the relationship between different metabolisms, and come up with an order in which they likely were invented. So that is all I am going to say right now on the antiquity of microbial life, and if you are interested, tune in for lecture three in this series, where I will spend some more detail talking about how we use a particular compound found in lipids in modern cells as a potential indicator for oxygenic photosynthesis and whether or not this is a valid thing to do.

The next point now I want to turn to is just how numerous microbes are. So let's ask a very simple question. How many microbes are there on Earth? And to bring this into a human reference point, let's begin with the number of the human population. So I am from Los Angeles, which at the latest census, was around 10 million people. And in the state of California, we are up to approximately 35 million, and in the United States in general, nearly 300 million. These are large numbers, but overall in the world we are up three orders of magnitude. at 6 billion people. And that's a lot of folks. However, this is nothing in comparison to the microbial population as estimated by a wonderful paper that I am citing here at the bottom of the slide called, "Prokaryotes, the Unseen Majority" that was published in PNAS in 1998. These are very rough numbers, but give or take an order of magnitude here or there, I think you are going to be impressed when you see the number that I am about to show you.

So the estimates for the microbial population are just enormous, 5 times 10 to the 30th cells. And this indeed is such a large number that it is very difficult to wrap our minds around it. So to try to make this a bit easier to do, I did a very simple calculation, where I assumed that the length of a given micro-organism was one micron and asked, "how many times would we need to go back and forth between the Earth and the Sun if we lined up all of these organisms end to end in order to account for this number?" And the answer, shockingly, is we would need to go back and forth 200 trillion times.

So hopefully that impresses you with just how many of these creatures there are on the planet. Now where are they, if there are so many? How come we don't think about this all the time? Why aren't we overwhelmed? Well, one reason is that oftentimes we are shockingly ignorant about the fact that they are all around us, that we ourselves are walking micro-organisms. So one of the first scientists to appreciate this profound fact was the father of microscopy, Antony van Leeuwenhoek. And this is a lovely image that he drew from his observations down his first microscope in 1684, and you can see he drew some nice rods and cocci, and even pictures of probably motility what is meant by these dotted lines from C to D. And he reflected, as he was looking through the microscope about his own teeth, and this is I think a very funny quote.

He said, "Though my teeth are kept usually very clean, nevertheless when I view them in a magnifying glass, I find growing between them white matter as thick as a wetted flower. The number of these animals in the scurf of a man's teeth, are so many that I believe they exceed the number of men in a kingdom." Well, this indeed is actually an underestimate. Not only do they exceed the number of men and women in a kingdom, they go far beyond that. So if we actually look at our own bodies… just take a look at your wrist, at one square inch on the surface of your wrist. Right there, we are estimated to have five to fifty thousand bacterial cells. And it just increases in density as we move to other parts of the body, such as the groin and the underarms, in our teeth, and really where it's mainly at in our bodies is in our colon.

And the overall total per person is seventy trillion. That is quite a lot. And one thing that I think is really important for you to know about the microbial community within your own body, is that there are ten times the number of microbial cells in our system than there are human cells. And not only that, when we look at the genetic potential of the DNA within these organisms, the genetic potential of only those within our guts is over one hundred times that of the human genome. So you might begin to ask whether or not humans are not merely walking vats of microorganisms, carriers serving their existence. It is something to think about, and there's a great deal of research now emerging that is beginning to illuminate just how crucial these organisms are for human health, not only with regard to being able to help us digest our food, but also interfacing and controlling our immune system, in ways that are fascinating and profound.

Now despite the fact that this number, ten to the twelfth, seems really large, and indeed it is, it's peanuts when we compare it to other domains where we find microorganisms. So let's start with the least abundant, up in the air, It is quite amazing to me that they've been detected as high as thirty four to forty six miles up into the sky. But these concentrations are really small relative to other compartments. As I told you, within the human body we have quite a few. And when you add up all of the humans and domestic animals, and then termites, which I'll get back to in just a bit, the order of magnitude jumps up to about ten to the 23rd, to 24th This is superceded by the quantities that you can find in soils, in forests and grasslands, deserts, tundras, swamp environments. These places are very fertile homes for microorganisms and there their activities can transform the chemistry of their environment quite profoundly. And this is of course also true in aquatic domains, where at similar orders of magnitude we find microorganisms in both marine and freshwater environments.

But all of these numbers pale in comparison to the numbers that we find in the subsurface, both in terrestrial and oceanic environments, where microorganisms have been detected as deep as two miles. Now, this really is a very interesting frontier area in microbiology It is hard to go down into these depths, and yet nowadays, researchers are equipped with the tools they need in order to access these remote communities. And what remains to be learned is what exactly these organisms are doing in situ. Are they active? And if so, what are their activities? Are these activities affecting in a significant way the physical and chemical properties of these environments? We don't know, and we look forward in the coming decades to finding the answers to these and other interesting questions. So now let me just give you an example, a tour through various parts of the world and other inhabitants of that world where we find these organisms. Just to bring home to you how ubiquitous microbes are on the planet. So to start with what might be a more familiar image, here what you are looking at is pond scum.

You are looking at a wonderful assemblage of phototrophs and other microorganisms in this pond. And my favorites of course are these purple phototrophs. These are the ones that I told you about earlier that are what we call the anoxygenic phototrophs that are not utilizing water as a substrate in photosynthesis, but are utilizing other more reduced compounds such as different types of sulfur species, hydrogen, or iron. Now these organisms that we see in modern day ponds, as I told you at the beginning when I was illustrating the antiquity of microbial life with the example of the banded iron formations, are absolutely historically important for their metabolism, and the diversity of their metabolism, and how it's changed the geochemistry of the Earth. Not only has the evolution of photosynthesis contributed to evolving our atmosphere to one that contains oxygen over the course of time, but as I also showed you with the banded iron formations, these types of organisms have likely shaped ore formation as well.

And many other important processes have been able to come about thanks to these organisms doing what they do, and it should be noted that this type of metabolic activity, photosynthesis, is one that today we are highly interested in because of our need for coming up with alternative energy sources, and certainly if chemists were able to mimic what these wonderful microbes in this pond do, we would be able to not worry so much about our dependence on foreign oil and our fossil fuel supplies being burnt, but that's a story for another day. The point is, their metabolic diversity is old. We see it all around us, and the biochemistry is really quite fascinating. Continuing on with the chemistry and the metabolism of these organisms not only do they do important things when they are growing, but they also do important things when they start hitting what we call stationary phase.

And this is a point in their development where they're not necessarily actively growing, but they are at a higher density and they are just hanging out metabolically. And when this occurs in their lifecycle, sometimes metabolites and pigments begin to be excreted. And these pigments, which are called secondary metabolites, although that name itself may be a bit misleading, because they are only secondary in a temporal sense, in that they are made after a phase of active growth, but by no means are they secondary in terms of the physiology of the organisms that produce them. None the less, these metabolites oftentimes are used today by pharmaceutical companies as natural products that confer antibiotic activity. And a terrific example of this are organisms in the Streptomycetes family that you see here in this Petri dish that are producing a whole host of wonderful antibiotic compounds.

Now containing in the environment of the soil of course are roots of plants. And in this part of soil known as the rhizosphere, we can find microorganisms as well that are colonizing in a very beneficial way the plant roots. And here is a tomato root seedling. This is an image taken by Guido Bloemberg. And he showed in experiments in the laboratory that when he took tomato root seedlings and mixed them with an organism called Pseudomonas, that this bacterium was able to colonize the plant and form what we call biofilms on the surface of the root. And this is just one example of organisms that interact with plants. There are many that fall into this category with different names. And the bottom line is that they have a very beneficial relationship with these plants, where sometimes they produce natural products that fend the plant off from fungal predators and so they serve as biocontrol agents.

Other times these organisms are capable of fixing molecular nitrogen into a usable form and essentially acting as a natural fertilizer. Now crawling around in not only soil environments, but of course we are very familiar with these from our homes, are termites. And the termites are a terrific source of microbial diversity and one that is becoming an increasingly important micro-environment in which to look because of our desire to understand microbial processes that might be harnessed for lignocellulose degradation. Again, out of a need to develop alternative sources of energy.

Now a colleague of mine, Professor Jared Leadbetter at Caltech studies these termites, and he likes to call them "an ecosystem in a microliter". And I think this is really a fantastic description of them because it is within their hindgut that you find a zoo of microorganisms and protozoa that are swimming around doing all sorts of important activities that make it possible for the termites to digest their wood. And in the process they emit methane, and not an insignificant fraction of this methane ultimately makes its way up into the atmosphere and contributes to the overall chemistry on the planet. So speaking of methanogens, here you see a dramatic illustration of them at work. This image that I am standing in front of is taken from Cedar Swamp in Woods Hole, Massachusetts. And it is an image from a group of students from the microbial diversity class, which is a fantastic course for about twenty students, half from the United States and half from overseas, who come together every summer to understand how microorganisms are able to perform these various metabolic activities that I have been describing in these lectures.

And what you can see here is that the students have gone waist deep into this swamp and they have stomped around, and as they have done this they have collected the bubbles that come up as they stomp the sediment, and collected them in these inverted funnels. And then some brave individual holds that funnel and removes their hand just at the moment when a friend comes by with a flame, and ignites it, and here you see a lovely illustration of methane at work. So methanogenesis led to the creation of the methane gas that was ignited here. Now in the past the activity of these organisms that generate this methane that are called methanogens, might have been important in shaping the chemistry of the Earth's environment.

And the reason we suspect this may be the case is because early in Earth's history the environment contained appreciably more methane than it does today. Now a different example of a habitat where microorganisms are very important is in Chile and in other places on Earth, but this example here is taken from the Andina Copper mine in the Andes in Chile where microorganisms are exploited for their abilities to help with bioleaching. And so what happens is that in these mines there are piles that are built up, and they are fertilized essentially with indigenous microbial populations that are able to live in shockingly low pH levels, down to pH as low as one, and sometimes even lower. And these organisms are essentially eating the minerals in this mine pile and the process of metabolizing it, changing the mineralogy, in such a way that copper is solubilized and leached. So here is another example of an environment that is quite extraordinary and yet microorganisms have been able to adapt and even to thrive in this extreme condition. So on our tour of extreme pHs, we just saw an example of low pH, so let's go to a high pH environment.

This one I am showing you is Mono Lake that is in Northern California. And Mono Lake is quite an extraordinary place. It looks almost like it is from another planet. You see these beautiful tufa towers that are calcium carbonate minerals forming, and it is because the pH is so high and the alkalinity is so high that they naturally precipitate from these waters. In addition to having these carbonate minerals, contained within this lake environment is a ton of arsenic, and I will get to this in part two of my lecture today. And what I want to point out right now is that in this very high pH environment, and also one that's replete with arsenic, nevertheless we find organisms called alkaliphiles that thrive here, that are able to make a living utilizing arsenic as a terminal electron receptor in respiration. This is the subject of my second lecture. And in so doing account for 14% of the carbon turnover in this system. Now let's go on to another example of an extreme environment. Here now we are looking at an extreme of salt.

And there is no better example of this than the Dead Sea in Israel, but you can find organisms such as those that inhabit the Dead Sea also in the Great Salt Lake, and other places on Earth such as salt flats, where you have very high salt content. And the organisms living here are capable of growing despite this high salt and have adapted particular molecular strategies to cope with it. One very elegant example of this is their ability to use special photopigments called rhodopsin and these are colored purple. And these rhodopsins, they have in their membranes, and enable them to generate energy under conditions where they need to use slightly different strategies than organisms that are growing under conditions that we would consider more normal. Now, so approaching the end of our tour through microbial diversity and ubiquity, I want to end with a few other extremes now that are based on temperature and pressure. If we think about the extremes of cold there is no better place to go than Antarctica.

And you might be surprised to realize that even in this environment you have microorganisms thriving in the crust. And these organisms are psychrophiles, and they're ability to grow is dependent upon dust from winds carrying nutrients picked up from the continents surrounding Antarctica, South America, Australia, Africa, that reach Antarctica, deposit their dust and fertilize these upper crusts of the ice where we have intrepid pioneer organisms that are able to utilize these nutrients and grow, even in these very cold regimes. So another extreme is that of temperature and pressure, and there is no better environment in which to observe this than at the bottom of the ocean, in environments where we have hydrothermal vents that are releasing nutrients into the deep. And here is an example of one of these vents.

It is called a black smoker because the nutrients that it releases, including manganese and iron, often precipitate in the conditions of the oceans at these sites such that they look black. Now around these vents there is abundant life, really extraordinary life, not just microbial life. but giant tube worms, and fish, and other macroscopic organisms. So the ability of all of this abundant life to be in this environment crucially depends upon the activities of microorganisms that are chemosynthetic. that are able to grow by the oxidation of sulfur and other compounds that you have present in this environment, and couple that oxidation of these reduced substrates to the fixation of CO2 into biomass. And this is at the base of the food chain that then sustains the growth of other marine organisms. such as these tube worms. And here you see an example of that in these beautiful tubeworms. If you cut them open and you look at one of their organs, called the trophosome within these organs are bacterial symbionts that are doing the process that I just mentioned.

So my final example that I will end with is one that might be the most familiar to you if you have ever done any PCR in molecular biology. So most of you have heard of the enzyme Taq polymerase, and this polymerase is what allows us to do an amplification reaction when we are doing PCR. Now this enzyme, Taq, derives from a bacterium called Thermus aquaticus, that is where the Taq comes from. The "T" is from the Thermus and the "aq" from aquaticus. And this is a thermophile that was isolated in Yellowstone at a hot spring, many decades ago. And it was presciently realized by Kary Mullis and others that the enzymes contained within it could be useful for various biotechnological applications because they wouldn't denature at the temperatures that would kill most other types of cells.

So these thermophiles are a very fascinating group of organisms whose molecular adaptations include not only DNA polymerases, but also a wide variety of other enzymes that might be of industrial use. So let's now end with diversity, which is really my favorite part of the microbial world. And I want to cover a few different areas of this. The first is phylogenetic diversity. Now one of the most important lessons to be learned in evolutionary theory was learned several decades ago from work by Carl Woese and his colleagues, including Norman Pace, who applied Carl Woese's fundamental insights into the diversity of life to the natural world.

And these individuals together with others were able to demonstrate very clearly that when we think about the diversity of life out there on the planet, we are really talking about a microbial world, whether we call these microorganisms Bacteria or Archaea or even Eucaryotes. What I want you to appreciate is that when you look at the tree of life, that's what this is. It is a tree that is drawn based upon comparing the sequences of a very particular molecule that every living organism has, that is ribosomal RNA, that is necessary for the process of translating messenger RNA into protein.

Because this is a very universal and highly conserved molecule Carl Woese and colleagues were able to deduce that it was a beautiful molecular chronometer that we can employ to look at the evolutionary relatedness between different organisms. And when he and his colleagues did this , he recognized that there were three primary domains of life, the Bacteria, the Archaea, and the Eucarya. And moreover, what I want to stress now is that our entire universe of Homo sapiens and humans and plants and animals, the macroscopic eucaryotic world, is only occupying in terms of this space on the tree, which is known as a phylogenetic tree, meaning a tree of evolutionary distances between different types of life forms, a very tiny miniscule branch.

And everything else that I am showing here is microbial. So hopefully that impresses you, but before we leave this tree let me point out two more facts that are very important. All of the metabolism on the planet was invented by microorganisms including the metabolism that we perform in our bodies today in our mitochondria. So the mitochondrion is nothing more than an ancient bacterial cell that invented the ability to do oxidative phosphorylation, which I'll tell you about in a little bit, that was engulfed or brought into symbiosis with some other type of cell, and over the course of time involved into the organelle that we call the mitochondrion. But it was a microorganism first, and that is where the beautiful metabolism that it goes through was generated. The same story is true for the chloroplast. This is nothing more than cyanobacteria that over time turned into plastids and became incorporated into other cells. Now the next important point I want to make is that microbial diversity also manifests itself morphologically. And this is something that only recently we are coming to appreciate in its full glory. Back in the days of Leeuwenhoek, when he had a simple microscope, all he could really see were different shapes of microbes, and to be quite honest, that is not terribly spectacular and includes rods and spirals and some cocci.

Once in a while you see higher structures forming of communities however, and Leeuwenhouk didn't necessarily know about these, but here is an example of one here. This is a beautiful example of fruiting bodies beginning to form by the soil organism Myxobacteria that does all sorts of interesting things when it comes together in a group that it wouldn't do as any individual cell. This is social behavior. So this is an example of bacteria acting in a multicellular fashion, if you will. Microorganisms, however, can get remarkably large. They are not just on the scale of microns. And here is a good example of this. This is, to my knowledge, one of the largest microbial cells known to date. It is called Thiomargarita namibiensis, which means the sulfur pearl of Namibia. And it is on the same scale as the eye of a fruit fly.

And when you look at it in more detail, the reason it is so big is that it contains this huge vacuole filled inside with nitrate, which is one of the substrates it uses to power its metabolism. And it couples the reduction of nitrate to a more reduced form of nitrogen to the oxidation of sulfide, and in this way it powers energy for growth. But let's leave the metabolism aside and stay focused now just on the form. Here is an example of one of my favorite organisms, Rhodopseudonomas palustris, and the reason I am showing you this is simply to illustrate that it has quite an amazing membrane structure within it. One that is reminiscent even of the Golgi in higher organisms. And indeed it might have been the progenitor of that at the cell biological level. And how these various structures form, these are what we call the inner cytoplasmic membranes where the photosynthetic machinery is housed in this case, in terms of the detail of what creates their shape is an open and exciting question that future microbial cell biologists will no doubt solve. But the final example, which is probably my all time favorite, is of an organism called a magnetotactic bacterium.

And here you see if you just look at it in a light microscope, although this is actually an image of fluorescence where we have put some GFP into the bug, it looks like just a common spiral. If you take a fancier microscope, a transmission electron micrograph, and cut it open and do a thin section, you can see that it has this beautiful chain of magnetic particles inside it. And now what I am going to show you is, I think, the best advertisement for the beauty of bacterial cell biology that I know, and it is a cryo-electron tomogram of one cell. And this was work done by Arash Komeili who is now a professor at UC Berkeley and his collaborator Zhuo Li in Grant Jensen's lab at Caltech. And together we made this movie showing the internal structure of these organisms.

So what you are going to see now is coming up through the bacteria different sections, and here you see the magnetosomes coming into view. Those are the membranes that contain the magnetite. If you missed them, now look, OK. Here they are in red, those magnetosome membranes, and then there is this yellow filament surrounding them. And what we have come to appreciate is that this filament is a protein that is very similar to actin. And it is necessary for these magnetosomes, for these organelle-like, although they never separate from the membrane, so they are not true organelles. Here you see they're attached by a neck that's only 5 nanometers in diameter, which is quite amazing, to this inner membrane.

They invaginate and form these vesicles within which a beautiful single domain crystal of magnetite can form. And this order, the fact that they are linear in a chain, is enabled by a cytoskeletal filament, an actin-like protein. OK. So the next to the last point that I want to make on diversity is behavioral diversity, and there is another lecture in this iBioSeminar series by Professor Bonnie Bassler from Princeton that can give you more information about this if you are interested. But what I wanted to point out here while we are going through a tour through diversity is simply that microorganisms can act in ways that are quite extraordinary when they are acting as a group. And you can see that illustrated by the activities of the bacterium Vibrio fischeri within the light organ of a squid. And here is an image that is from the beautiful pioneering work of Margaret Mefal-Ngai and Ned Ruby at the University of Wisconsin, Madison where they have been studying for decades the interactions between the microorganisms in the light organ of the squid and the squid, and the ability of these organisms to colonize this environment, and when the lights go out at night, emit a beautiful luminescence.

Here you can see pictures of these organisms that have just been streaked out on a plate in the dark. They are glowing. Well, they glow as well here at night in the belly of the squid. And it shields these squids from predators below because the light of the moonlight coming down from the top is roughly of the same luminescence as the light that they are emitting. So it allows them to have a stealth function and glide around in the oceans and be unseen to predators deeper below them. Now, this isn't just a phenomenon that affects the squid. This is a phenomenon that can get quite enormous in its scope. And the best example to illustrate this is this satellite image here taken off of the Somalian coast, where you see an image that quite literally is of milky seas as described by the ancient mariners, but what today we understand as glowing bacteria.

In this case an organism, likely called Vibrio harveyi, associating with micro-algae in this environment that for whatever reasons that are not fully understood at this particular point in time when this satellite image was taken, had a bloom and began luminescing like crazy, and filled up a volume the size of Connecticut. All right, so, to end I want to just mention a few rules of microbial diversity because almost everything that I have talked about so far in this lecture ultimately comes back to the ability of organisms to generate energy in ways that are quite amazing. And I would stipulate that microbes are by far the best chemists on the planet. And so if you are a chemist, pay attention, because a lot of lessons can be learned from these guys. All right. Now when we are talking about the phase of active growth, the bottom-line that microbes are facing is they simply want to divide. And to do this they need two things. They need energy, and they need carbon. And beyond that, they are virtually unconstrained, although there are a few constraints, and we will come back to that in a moment.

They need substrates, and these substrates can be organic or inorganic compounds. This is now for the part where they are going to be generating energy. Those substrates are converted to products through catabolic reactions, or energy generation, if you will. And often times we think of energy generation in the form of ATP, the most important energy carrying molecule within the cell. Now this part of metabolism, catabolism, is coupled to anabolism, which is the part of metabolism that is concerned with energy consumption, or biosynthesis. And now down here what we are talking about is the conversion of carbon, often in the monomeric form, to biomolecules that are far more complex, so protein, DNA, lipid, for example. Now if we are thinking just about the substrates, as I said they can come from a variety of sources.

Always they're chemical, although light can help enable cells to actually utilize those chemicals in ways that they otherwise wouldn't be able to do. But when we are talking about the growth of organisms just purely on chemicals, without needing a boost from light, the name we give to this metabolism is chemotrophy. And that in turn is classified into two different types, inorganic and organic. And when we are talking about inorganic sources of energy like hydrogen, and sulfide, and iron minerals, this is called chemolithotrophy. And when we are talking about growing on organic substrates like glucose, or glycerol, or acetate, this is called chemoorganotrophy.

And of course, as I said, while chemistry is always at the basis for any type of metabolism, there is a photochemical boost that is often necessary, when activating a compound that otherwise might not be biologically utilizable for energy, and that is when we call that process a phototrophic one. So the final part of this that I want to just mention is that the carbon source, which is distinct or can be distinct from the energy source… sometimes they are the same thing, but they don't have to be the same thing… is either coming from inorganic carbon, CO2, or organic carbon. And when it is coming from inorganic carbon that is called autotrophy, and when it is coming from organic carbon, that is called heterotrophy. So we are heterotrophs, we need to eat some type of organic carbon whether we are vegetarians or meat eaters, but microorganisms are far more sophisticated.

They can eat minerals. They can just take CO2 from the air, and they'll be on their way. So finally the last part I want to mention about metabolic diversity writ quite large is that you can generate ATP through one of two different ways. The first way is through what is called substrate level phosphorylation. And this is also termed fermentation, and essentially is the process where different types of reactions between chemicals within a cell enable transfer of an inorganic phosphate ultimately to ADP to produce ATP. And this process is enabled by chemical rearrangements within the cell and reactions one on one between compounds. The next major way that ATP can be formed in a cell is through the remarkable process of oxidative phosphorylation. Basically this is about electron transport chains in membranes that are coupled to generating a battery around a membrane by extruding protons to one side and polarizing it so that there is an electrochemical potential gradient across this membrane that can be harnessed to do the work of making ATP.

Now something that I am not showing you on this diagram, but I want to introduce as terms are an electron donor and an electron acceptor. So in metabolism there is always a substrate that is used as the primary electron donor that can be metabolized through various pathways and reduced to a compound that can donate electrons to the electron transport chain in the membrane. And then there is always something that serves as the acceptor of those electrons at the end of the chain, and that is called the terminal electron acceptor. And it is the path of electron transfer and proton translocation between this electron donor and this terminal electron acceptor that is really harnessed by the membrane to do work. And so that is what you see here pictured very generically without a whole lot of detail in the sense that through this electron transport process, which imagine if you will, is coupled as I said to proton translocation, and that is achieved by different things within this membrane.

They can be proteins, or small molecules that are able to simultaneously pass electrons through the membrane to something else in the electron transport chain and push protons, or translocate protons across the membrane so that there is this gradient that arises where there is more positive charge on the outside than on the inside. Now once this happens this gradient can be used to drive ATP synthesis. And this happens through a really amazing molecular machine called the ATP synthase, which allows the traversal through the membrane of a proton, that concomitantly gives the energy to phosphorylate ADP, adding that inorganic phosphate on, and making ATP.

And as this happens, the electrochemical potential gradient lessens. And so that is what I am showing here: the energized membrane due to proton transport coupled to electron transfer through the membrane, and then this being expended and used in order to drive ATP synthesis. Now while you can imagine a whole variety of things that can be electron donors and electron acceptors from microbial metabolism, metabolic diversity does have to conform to some rules. And there are three that I want to point out that I think are particularly important. The first is that the amount of energy has to be at a very minimal level, at least in order to sustain the cell, both with regard to active growth, where you need a high level of energy, at some threshold amount in order to double, but also at the level where you are generating enough energy simply to maintain basic cellular processes even if they are not coupled directly to growth.

Now what is this number and how do we constrain it? Thermodynamically, this can be expressed in this very straight forward equation here, which is saying that the standard free energy that can be gained from a process where there is electron transfer between the electron donor and the electron acceptor is a function of the number of electrons transferred, multiplied by the Faraday constant, and this in turn multiplied by the difference in redox potential between the electron donor and the electron acceptor.

So for example, a common intracellular reductant, is NADH, and in its oxidized state this is NAD+. The redox potential of this redox pair is very low. It is very negative on the electron potential scale that is typically expressed in millivolts. On the other side of this scale are the electron acceptors with very high redox potentials, like oxygen. And so when you couple through the membrane a process of electron transfer from NADH to oxygen, thermodynamically you have the potential to generate a lot of energy, and this is captured through a beautiful sequence of proton carriers and electron transfer biomolecules contained within these membranes. But these electron transport chains need not be between NADH and oxygen, you can have a whole assemblage of things that can interrelate and so the minimum amount of energy that needs to be supplied has been calculated. And this is a very crude estimation, but it is an interesting study, and I refer you to below where you can see the reference. Where for organisms operating in very low energy regime, it was inferred that the minimum free energy required to sustain them and their growth was about -4 kilojoules per mole, and that is about as low as you can go at least as experimentally measured.

Finally, regardless of the thermodynamic potential there are two other very important factors to keep in mind. The second point is that the substrates themselves must be bioavailable. And so this is more of a kinetic problem where we need to consider accessibility and transport of substrates across the membrane to the site in the cell where they are used. Or the ability of the cell to figure out a way to access them even if they can't transport them inside. And the final point is that these substrates or the products after the metabolism has done its thing must not themselves be toxic.

So in the next couple of sections of this lecture, I am going to give you examples of different microbial metabolisms to illustrate these general points I have been making, but I hope what you will remember from this seminar is the four big points about microbial diversity. One, that it is incredibly ancient and over this long period of Earth history numerous microorganisms in ubiquitous environments have evolved diverse metabolisms that allow them to catalyze fascinating chemical reactions and that these reactions have affected not only the ability of the cells to grow and divide, but in many instances have profoundly affected their environment, be that environment one in an ancient ocean, or today inside the human body.

Thank you..

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