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Unit 4: Microbial Diversity
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Anna-Louise Reysenbach, PhD

Anna-Louise Reysenbach, Ph.D.
Interview with Anna-Louise Reysenbach, PhD Reysenbach is a microbial ecologist with special interests in the ecology of terrestrial and deep-sea hydrothermal vents, and in the evolution of biogeochemical cycles. She teaches the core microbiology course at Portland State University, as well as courses in Microbial Ecology and Microbial Diversity.

Yellowstone is incredible, what makes this place so special?

There are many reasons why Yellowstone's special for me personally, but also professionally. I will stick to the professional. Yellowstone-the thermal features and the microbes associated with it-provides an incredible natural laboratory for research.

For an ecologist it's even greater because you have variations in temperature, pH, light energy, and geo-chemical energy. The pH's can vary from zero to nine, so that whole range of pH's and whole range of temperatures provide all these differentenvironments for a huge diversity of microbes. From that perspective, it just provides an incredible model systems for studying different microbial questions. Since I'm an ecologist, I am interested in the dynamics of communities as [they are] associated with chemistry. So it's perfect-you just walk up to a spring and you can be able to do your experiments right there.

How much do we know about the microbes here versus how much do we need to know about them?

It's been estimated that in the microbial world we've probably only described about one percent of the diversity. There is a huge diversity in the natural microbial world that hasn't been described and we have now the techniques to be able to describe those organisms. Using those techniques, there have been some very big surprises, particularly here in Yellowstone.

[In] almost every sample we take we discover new organisms that have never been described before. It's important not only from a science point of view but also [this] diversity is equivalent to genetic diversity, [which is] equivalent to biodiversity. Preserving biodiversity and genes in particular is important to the survival of this planet, in a way.

Is the diversity of microbes greater than the diversity of plants and animals?

Yes, the diversity of microbes is greater than the diversity of all plants and animals put together and probably much greater. We've only just started to scratch that surface of what is the diversity of the microbial world.

Is that true as well in Yellowstone? There is more diversity in the microbes then all the plants?

Yes, so when [we] look at a microbial mat [and] describe all the diversity in the that we looked at, there is probably more diversity in that little square then there is in terms of the visible diversity in Yellowstone, which is amazing. That is pretty spectacular.

You have categorized this place as an analog to early Earth? What does that mean?

Environments like Yellowstone are considered [to be] good model systems to study early Earth processes. They could be analogs to what life was like on early Earth.

The reason why they could be good analogs for such studies [is] when early Earth formed, it was a very hot fiery planet. Then slowly but surely, Earth cooled down to the Earth we know today. So these thermal features may be relics or fossils from what it was like on early Earth.

Understanding the types of processes that occur in these systems may help us understand what it was really like 3.5 billion years ago.

How do you know thermophiles are at the base of the tree of life?

We don't absolutely know. We use mathematical models and comparisons of genes of you, myself, the bison, and all these other genes. We are comparing the same gene that we all have and it's a conserved gene. It's been around since the beginning of life. We use mathematical models to see how [many] changes have occurred through time. The tree of life is still a model, we can actually figure out that some of the more ancient genes belongs to these ones.

It sounds like the microbes were driving their own evolution-you havetermed them master chemists.

That's a really nice way of saying it. I often consider microbes the chemists of the world. Chemolithoautotrophes are the best chemists because they just use the chemistry and make some other chemistry. They take one form of iron and make another form of iron. In a way they can actually change their very little local environment. In the evolution of life, they really have changed our atmosphere because evolving oxygen was a microbial process. It totally changed Earth as we know it today.

What kind of lineages are you working with and why did you choose to work with them?

Most of my research in the park has focused on a group of organisms that is one of these deep lineages within the tree of life. They are called the aquificales. "Aquif" means water-maker. The reason why I've been very interested in them is that they are extremely dominant. [They are] the main organism in some of these hot spring environments, mainly the ones that are near neutral (near pH 7). You don't really find them as commonly in the low pH's. A hot spring like the one here at octopus [is a] high temperature environment (anywhere from 80 to 65 degrees Celsius). [If] you see some filamentous biomass, something that looks living, it's going to be these aquificales, that's very unusual. It makes them very interesting to study. It's only veryrecently that we were able to identify these organisms and what they were doing.

The focus of my research is actually to identify them: then try and figure out what exactly are they doing in the environment, what energy sources are they using. Because hydrogen is not the only energy source. There is a lot of iron with oxygen, a bunch of different combinations of electrons and acceptors which is how you get your energy. Once I know what they're doing, [I] try to figure out how to grow them in the lab. [Their] biological activity will eventually be recorded into rocks and so you can actually begin to look back into the rock record, when these organisms may havebeen active billions of years ago.

In some of these hot springs they are the most dominant organism. They are almost the only thing there. They are like a mono culture of organisms, a wheat field in a little space. We know very little about them so it's interesting to know why they are so successful at these very high temperatures. And thirdly, the aquificales are involved in mineral precipitation. They take out of solution metals like iron or sulfur. These little filaments are covered in iron, just little black filaments. At another site, they are covered with sulfur-they sort of have this white, yellow color. From a geological perspective that's interesting, because what happens is as these organisms die and dry up and the water moves in a different direction, that which was once biologically active now becomes mineralized and fossilized and becomes rock.

I mentioned that some of these organisms are the deepest lineages in the tree of life. The way we look at life at 3.5 billion years ago is we look at the rocks and say "OK, there was life at 3.5 billion years." The way life has been shown to be at 3.5 billion years [is] to look at fossils. They are micro-fossils, tiny little blobs in the rock record.

There are other ways of looking at [the biological record]. They are called isotopic methods [in which] you look at an isotope. Something like carbon as two different isotopes (it actually has three). It has the radioactive isotope, but then it has a stable isotope. There is a carbon 13 and a carbon 12. For example microbes, biological systems, prefer the carbon 12 over the carbon 13. If you give a microbe carbon 13 carbon dioxide or carbon 12 carbon dioxide, it will preferentially take up the C12 carbon dioxide. When its biomass gets fossilized in the rock record what you'llsee is rock that has a high concentration of carbon 12. You know then that [the] rock has biology in it. So those are the two major forms of showing [that] life existed 3.5 billion years ago, micro-fossils and biological signatures like isotopic.

Can you talk more about micro-fossils. What did they look like? What are the shapes of these fossils compared to the ones now?

A lot of these fossils that are in early rock records are long filamentous organisms, very similar to the kinds of things we are looking at here. They are also very similar to some cyanobacteria. So it's very hard to distinguish whether they are chemolithoautotrophs or photosynthetic organisms. We don't know what those original fossils were.

You study particular organisms in more than a terrestrial environment. Where else do you study them and why do you study both?

The other very exciting thing I find about having focused my research on this group of organisms is that we found them at deep sea hydrothermal vents as well. So they're global. They are everywhere where there is a high temperature environment. We've discovered a whole new lineage within the aquificales, which [is] closely related [to] the ones I work with at Calside Springs.

So they're both in the deep sea vents and terrestrial systems. It's interesting to know how do these organisms differ because those are very different environments in a way. One has a huge amount of sulfate and nitrate and energy sources are very different here in the hot springs. We are interested in looking at both environments. That will help tell us a lot more about the [organisms'] physiology [and] metabolism. Deep sea vents have these beautiful big chimney structures where we isolated [the organisms]. They too are associated with minerals.

I am interested in knowing how they affect the mineralogy, how they change the chemistry of their little environments. Both systems have good environments for studying those kinds of questions, how they change their chemical environment.

When you look under a microscope what do they look like?

A lot of these vary in size and shape-the pink filaments for example, are much longer filaments. The narrow filaments actually form little rods that form into filaments. Some of the organisms in the deep sea are little short rods. The ones at Calside are sort of fat little chubby rods and they basically stick together and make these filamentous mats.

What are you currently working on?

One of the projects I have in the park at Calside Springs [is to study] these incredibly beautiful, black, filamentous organisms. We've been studying them for quite awhile trying to figure out what kind of energy sources they are using. Why are they precipitating all this iron, etc. Using molecular methods, I was able to identify organisms as a separate branch within the aquificales related to these pink filaments, but not in the same branch. They are not of the exact same family.

At the same time I was working at deep sea vents and I found them there, too. [They are] very close relative of the ones at Calside Springs. We discovered them but all we had was a [single] gene sequence. We had no idea about them otherwise. To understand more about an organism, you really want to have its entire genome. One way to get that is to try and grow them in the lab. We were first able to grow those organisms in the lab from samples from deep sea vents. We recently named that organism Persephonella marina.

Which organisms have you sequenced?

We have these organisms in culture here at Octopus spring with the pink filaments, called Thermochrinus ruber. Because this group is so interesting, recently the NSF has awarded us a grant to sequence the genomes of both a terrestrial relative which is the one that is very close to the organism in Calside Springs and to sequence an entire genome of organism we got from deep sea vents. So this way we can compare the entire genome rather than a single little gene.

Why is that important?

That is really important because that will tell us more about not only the entire genetic information but it's capabilities. It may also tell us about what are some of the genes that have existed for a long time. The genome of an organism is its history. We're hoping that by comparing these two genomes we'll get some information about a lot of these questions. The one other close relative of T. ruber has been sequenced. That [organism] is called Aquifex pyrophilus. So we'll have one genome in this branch, this close relative of T. ruber. Then we'll have two genomes with the organisms I was talking about so that's pretty exciting.

Have you been commissioned to do an inventory of selected areas?

[We are] developing a microbial survey of a lot of the thermal features in Yellowstone. We're going to first focus on thermal features. Basically there has been no specific database of all the microbes [in] these hot springs. We're going to start that basic database and ten years from now, when you come to Octopus you can say, well there is no pink filaments now. This database [will have] microbial sequences and cultures, etc. My part of that project is to also help develop some of the educational components. Part of that is to teach a course in Yellowstone to educate the general public on Yellowstone's microbes. This will help the park in establishing educational booklets.

Why is it important for Yellowstone to know what they have?

It's really important for the park to have such an inventory because unless you know what you have you can't argue why somebody can't take something out of the park. Also, a lot of these microbes do have potential biotechnological significance and could have a major impact on biotech.

By doing an inventory of Yellowstone's microbes, it's sort of analogous to what type of biodiversity is in the Amazon rainforest. In that way we have a responsibility to know what kind of biodiversity we have on this planet. Microbial diversity has really lagged behind macro biological diversity. So in understanding what we have today, we will be much more responsible about managing our resources. It's important to this planet that we maintain it. A lot of [our genetic diversity] is microbial and until we really understand what we really have, we're really behind in managing biodiversity.

How is this helping us to determine whether there could be life on other planets?

Another reason for studying these types of environments is that they are potential models for understanding whether there could be life on other planets. The way wecan do that is [by] understanding that the extremes that these organisms live at could be the kinds of extremes that exist on planets like Mars. We brought a rock back from Mars, now what do we look for in the rock record? What are those signatures of life to look for? On Mars there is evidence for some volcanic activity. The types of organisms that live in these hot springs may have existed on Mars many years ago. So understanding how the processes occur in these environments may help us understand what to look for, or what environments to look for, for present life.

A lot of the research that has occurred in some of these extreme environments has opened peoples' minds to the existence of life [on other planets], wherever there is an energy source there may be life. So it really has opened this whole new field of astrobiology: Life on Earth and how it relates to other planets. There is a definite resurgence of understanding of microbial processes as they relate to processes on other planets.

Why are there different colors in the pools at Yellowstone?

The one thing that's really amazing about Yellowstone is that all the different colors of the different pools do reflect huge chemical and temperature gradients, so each pool's going to be very different in temp, and pH, chemistry. You can get pH from 1 all the way to pH 10. Temperatures from 50 degrees Celsius all the way to 93 degree Celsius so there's an extreme and a huge range of variables and therefore a huge range of environmental conditions that which different microbes can live in. And each microbe will be defined by the environmental conditions that they're in.

A lot of the colors that you see can either be minerals-[red means] a lot of iron, or the white, limestone. It's got some carbonate that's been precipitated. Some of it's mineral, but then some of the colors are microbial mats and a lot of those are greens or reds or pinks, and those colors reflect photosynthetic organisms a lot of the time.

The pink filaments are pink because they produce a carotenoid: it's sort of like the pigment that's in a carrot. We don't know why they really produce that pigment.

The other organisms that I work with are called "black filaments" and they're black because they precipitate huge amounts of minerals on the outside and they're iron minerals and so they're just black. There's another group of organisms that are yellow and all related to T. ruber, to the pink filaments, and they're yellow because they precipitate sulfur.

Does the color have something to do with how they make a living?

Carotenoids are produced by photosynthetic microbes to protect the photosynthetic machinery from excessive light and so the carotenoids could be used for some sort of protection mechanism. What the mechanism is I don't know.

Do the bacteria here form communities of organisms?

In a matter, it's like a symbiosis. The photosynthetic organisms are your primary producers, but some of them are aerobic primary producers; some are anaerobic primary producers. When there's no oxygen, they can still produce, use light energy. When there is oxygen, they can still use light energy so there's a lot of primary production going on at that level. They then supply carbohydrates for the heterotrophs, for the next level down, and so there's a lot of communication.

In the mats at Octopus, in addition to giving each other food, basically, they also create some structure so one group of organisms actually creates the mat structure in which the other organisms can live. They're held in there, otherwise they would just flow out. There are some filamentous organisms that really cause the structure and then these other organisms just sit in there in that matrix. It creates the environmental conditions, a little house for those other organisms.

Why don't they just keep growing?

They will not just keep growing and growing because they'll be limited by light, for one thing, so the top layers are ones that are going to be able to photosynthesize, and so eventually the ones down below are going to decompose, and the matrix is going to break down; you'll get a certain height but it won't just keep growing.

Do microbes help decompose the mat?

Probably at the base of the mat, mainly, at the bottom where there's not enough light. Mostly the organisms there are decomposers, so they're breaking down the mat using the organic carbon for their own growth. A lot of those are going to be anaerobic organisms that cannot use oxygen.

What is the difference between thermophiles and extreme thermophiles?

There's a pretty superficial delineation between thermophiles and extreme thermophiles or they're called "hyperthermophiles." A thermophile is an organism that grows between 45 and about 80 degrees Celsius. A hyperthermophile is an organism that grows best above 80 degrees Celsius.

So T. ruber would be referred to as a hyperthermophile whereas the black filaments that we work with in Yellowstone grows best at 70 degrees so it is actually a thermophile.

What did you do with the samples you collected before and what do you do with them now?

Until fairly recently probably about 20 years ago, the only way that we could identify a microbe is [by looking] under the microscope. You see this community of organisms, you see a little blob and a little rod and a little blob and another little rod and a little blob.

How do you distinguish between organisms that have no physical shape differences. The only way that we could do that until very recently was to try and grow those organisms from the environment. But just try to second guess what these organisms like to eat from the environment. Do they want carbon dioxide? It's really hard to second guess what these organisms require and they're very complex communities, like the mat they're feeding each other, so they're communicating with each other what they need.

To try and figure out how to grow them is not a trivial process and so, consequently we've only been actually able to grow less than perhaps 1% of all the organisms you see under the microscope. And that's just because we've been very selective.

What are the new tools you use today for identification?

The bottom line is until recently microbiologists couldn't be like biologists. Biologists can go into the field and they can identify a bison or a fireweed. We couldn't. But now we can. New tools have been developed, they're molecular based tools that enable us to go into the environment and actually identify the organisms. These are based on a diagnostic gene within the genome of each organism that we share with all microbes. Everybody has this gene called the small subunit ribosomal RNA gene. It's part of the ribosome. The ribosome is essential for protein synthesis and we all need proteins. Protein synthesis has been around since the beginning of life and so this gene that is part of the ribosome is probably evolutionary conserved. [This gene] enables us to relate different organisms to each other based on the gene sequence.

How was this gene discovered?

Through the work of Carl Woese who had been doing a lot of work on ribosomes and understood these basic evolutionary mechanisms of protein synthesis. Why [did] he chose this specific gene? He originally started with a smaller gene, but that had less information than this gene. These are all essential parts of protein synthesis, a part of the ribosomes.

There's another one that's called a 23S. The one we're working with is called the 16S and then there's another one called 5S. Each one is bigger and bigger-the bigger the gene is the more information it has. The more information you have, the more data you have to compare different organisms so you get a much finer resolution of the relationships of how many changes have occurred of evolution.

Can you grow T. ruber in the lab?

We can now grow T. ruber in the lab and that was actually based on first going to the environment, figuring out what those pink filaments were using the 16S ribosomal RNA gene. The way we do that is to go to the environment, we get the community and extract the DNA from all the different genomes of that community. We then obtain the small subunit ribosomal RNA gene-the 16S ribosomal RNA gene-from each of those different organisms in that community. We then sequence that gene and by comparing that gene with all the other organisms in the database, we're able to place that organism in a family tree of life. We were able to show that the pink filaments were closely related to another organism called aquaficales and this uses hydrogen and oxygen and carbon dioxide to grow. Then we try to grow them under those conditions and sure enough they were grown in the lab under those conditions without the 16S.

How did you isolate the 16S rRNA?

We extract the DNA from the sample from Yellowstone. [We] break open the cells using detergents or some sort of physical means-the cells just pop open and you separate the DNA from the proteins. Now you've got DNA from all these different organisms. Then you use a Polymerase Chain Reaction (PCR) to actually find the 16S ribosomal RNA gene in the genome.

The way you do that is you use little tags or little molecules that can actually find it. They basically bind to the DNA on either end of the gene and then you use PCR which uses an enzyme called "tac polymerase." It makes many, many copies of that single gene. What was once basically a needle in a haystack now suddenly becomes this huge amplified single gene in your test tube.

Is this sample the genes of just one organism?

No, you've got the 16S genes from all these different organisms. You've got the 16S from this little rod bacterium and you've got the 16S from this little round bacterium, you've got this little 16S from the little squiggly one and they're all in the same tube.

Now you have to separate out those genes because what you want to be able to [do is] sequence that information. [This] information is then used to generate this big tree.

You separate them out using a bunch of different techniques. One of them is cloning and the other one is called gel electrophoresis and it separates out the genes on a gel. You can actually see the genes on a gel and you know that each gene is from a different organism.

Once you've separated them out with whatever method you sorted with, you then obtain the DNA sequence of that gene, and that's the identity. That's the fingerprint of that organism.

The fingerprint is the sequence?

Yes, it's the identifier. Once you have that DNA sequence, you've got the identity of the organism. When you [have] 1,300 or 1,400 bits of information, which is the DNA sequence, you can actually find parts of that DNA sequence that are absolutely unique to that organism. Then you can design another piece of DNA based on that information-something called a "probe" -and you can make a piece of DNA, the machines will do that for you.

You add a fluorescent tag to that and you go back to the environment. The bottom line is you don't know where these DNA sequences come from. So you want to say, "this unique sequence that I have that's called EM17-where did that gene come from? Which organism?

The way we do that is something called "hybridization." You make the cells permeable and this little probe with the fluorescent marker goes into the cell. It finds its match because there are lots of ribosomes (lots of 16S RNA molecules). When it sees its match it binds and it won't bind to organisms where there's no match. It will then light up.

What are you looking for when comparing organisms?

When we're comparing these different sequences, we're looking for differences between the organisms. The more differences there are in the sequence, the more different the organisms are, the more they've diverged from each other. The less differences, the more similar they are to each other, and so that will show up in the family tree. There will be short branches and there will be closely related and those have less differences than the longer branches.

How does this help us know about evolutionary differences?

There are a number of computer programs that enable us to convert this difference in the nucleotides into an evolutionary difference. Those methods enable us to actually generate this evolutionary tree of life.

How has the tree of life changed with regards to this new technology?

We now know for a fact that there aren't five kingdoms of life, we know-and it's very well established-that there are three domains of life, the bacteria, the archaea, and the eukaryotes. The bacteria and the archaea are the prokaryotes. They're both organisms without a cell nucleus. They're as different from each other in this tree of life as we are from them.

What are the differences between bacteria and archaea?

There are numerous differences between archaea and bacteria. Most of the differences are based on chemicals. For example, the cell wall structure is very different. In the cell wall, all bacteria have peptidoglycans whereas the archaea do not have that. They have very different RNA polymerases. They have different ways in which they do some of the basic functions of the cell. They share with each other the basic morphology in terms of being simple, single celled organisms. They share a lot of other kinds of basic genes.

Are archaea more closely related to eukaryotes or bacteria?

Archaea are probably more closely related to us than we are to bacteria. Archaea are really good model systems to study some of the basic functions of our own cells, things like translation and transcription. There are a lot of similarities, and so a lot of studies are done on archaea because they're very easy to manipulate compared to eukaryotic cells.

Why is microbial diversity so vast?

There's a huge microbial diversity because microbes have developed the ability to use almost every energy source that there is available on Earth-where there's energy there will be a microbe. Eukaryotes have a much more limited ability to use different energy sources, basically light and organic carbon and that's about it.

What are the big surprises in your research?

The big thing that drives my research is that whenever we come back from a field trip, we always come back with big surprises, brand new branches of life that we never realized existed. What are they? How are they making a living out of those conditions? Those are the [questions] that drive me to try to figure that out.

The other thing is [that] a lot of the time we don't understand the communication between microbes. [We need] to figure out what are the interactions between different kinds of microbes and how do they influence each other, i.e., social behavior. A lot of time microbiologists try to separate organisms as single isolates but I think that's actually caused us to ignore some of the more important organisms because they need each other. [As] an example, there is one organism-a thermophile- that you can grow only if you grind up another thermophile: there's something the other thermophile's producing that this thermophile needs.

The average person on the street is going to ask why you care so much about what microbes do for a living and why should we care?

Because if you took them away you wouldn't be here. This is one of the big questions that I'd want my students to be thinking about. I want them to be challenged by questions, so a lot of the time I will feed them with something I am very interestedin and see where they go with that. I always hope they will go beyond what I'm thinking. I am hoping they are going to ask me questions that I don't know answers for because that's going to challenge me.

Actually that's part of the fun thing of being a professor: you have students who are continually challenging your knowledge. But also your wonderment of nature because they'll see things. I see things in a certain way even though I try not to be myopic and colleagues and students in particular can come and see things at a very different angle. That's what makes science so exciting, because you've got different ideas from different angles sort of congealing in a very big picture.

So what are some of the unanswered questions in this field?

Some of the real big unanswered questions are, how did life originate? We really have no idea. We don't know how early life evolved, really. What the steps are, whether thermophiles came first or whether chemolithoautrophs or heterotrophs werefirst. Those are huge questions.


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