Mitch Sogin heads the NASA Astrobiology Institute’s Marine Biological Laboratory team. At a recent conference, Sogin gave a talk about the work that molecular evolutionists do and how it has contributed to understanding the history of life on Earth. In this third and final part of a three-part series, Sogin describes the challenges that face researchers in their efforts to explore microbial diversity in extreme environments.
Microbiologists are interested in studying extreme environments to find out what organisms are living there, how diverse they are. Diversity is important for us to keep in mind in the context of planetary exploration.
But until recently, studying microbial diversity has been very difficult to do. One problem we face is that morphology (the form and structure of the organisms) doesn’t give you any kind of a metric for diversity. The second problem is that most microbes are difficult to grow in the laboratory. We can only grow one percent or less of the organisms in an environment.
For example, until about 20 years ago, marine biologists thought that there were roughly 100 organisms per milliliter of sea water, because that’s approximately what they could get to grow. John Hobbie started to use DAPI stains (which stain nucleic acids with fluorescent dye) and demonstrated that microbial life in the oceans was much, much more dense. When you use a DAPI stain on a sample of marine water and then look at it under a microscope, you typically see on the order of 50 microbes in your field of view. To see that many microbes in a single field means that there have to be somewhere between 10 thousand and 100 thousand cells per milliliter. But we can only cultivate about 100 of those organisms.
So cultivation has been a big problem, especially if you want to ask questions such as, “Is there evidence of novel evolutionary lineages when you look in an extreme environment?” or “How many different microorganisms are present?”
Of all the science that NASA’s funded in the area of biology, Carl Woese’s research was probably the most significant contribution. Because it provided a window into the microbial world, a quantitative window, not only in terms of assessing diversity, but also in understanding how microbes are related to each other. Woese also identified the Archaea as one of the primary domains of life. Woese’s insight was based on structural studies of ribosomal RNA. Ribosomal RNAs are present in all cellular-based organisms on the planet. By comparing ribosomal RNA sequences from different organisms, one can infer their evolutionary relationships.
If you want to look at an environment and ask the question, “What kinds of organisms are there?” you can use the presence of a particular ribosomal RNA as a proxy for the presence of a particular organism. You can also look at differences among the ribosomal RNA sequences. And then by using appropriate computer algorithms you can generate evolutionary trees that show you how closely the different organisms are related.
In 1987, based on comparisons of ribosomal RNA sequences solely from organisms he was able to culture, Woese had identified about a dozen different phyla (major groupings) of bacteria. By 1997, using molecular characterizations of DNA extracted from natural habitats, Norman Pace was able to enumerate 24 different major lineages, almost a third of which had no cultured representatives.. Today, about 80 different bacterial divisions have been identified and we believe that as we continue to explore new environments the number of major divisions is going to increase.
I want to talk about two sites where we’ve worked on this question of diversity. The first is Guaymas Basin, which is in the Gulf of California. Guaymas is one of a number of deep-sea hydrothermal vents that has been studied. Interestingly enough, microbial population studies of vents are actually quite limited: We only have detailed descriptions on half a dozen vent sites. It’s a very rich in terms of both bacterial and archaeal diversity. The surface of the Guaymas sediments is composed of a bacterial mat of filamentous Beggiatoa.
The system can be quite hot. One can extract cores close to the vents that range in temperature from just a few degrees C (about 40 degrees F) all the way up to 180 degrees C (about 360 degrees F). The deeper the core sample, the warmer it gets, to the point that it’s downright hot.
Among the archaea found at Guaymas, one sees methanogens (organisms that make methane as a byproduct of their metabolism), thermophiles, all the players that we would expect to find in this kind of environment. There is archaeal diversity. But of the 80 known bacterial phyla, we only see about a dozen in Guaymas. So it’s a moderately complex environment, but it doesn’t appear that all the lineages in the bacterial world are present.
It’s an anaerobic (oxygen-free) environment principally, in which there’s a lot of interaction between archaea and bacteria. The end result is that the system as a whole performs anaerobic methane oxidation. But it’s important to understand that this anaerobic methane oxidation doesn’t occur because of a single organism. Rather, it happens because there is a consortium of organisms. It’s really a very complex system. Indeed, complexity is an underlying theme for life in extreme environments.
Another reason we were interested in Guaymas had to do with eukaryotic diversity. We find many RNA sequences in these anoxic environments for eukaryotes that represent new lineages, novel diversity. And while the bacterial diversity only covers roughly a quarter to an eighth of the total diversity in the bacterial domain, almost all known kinds of eukaryotes are found in this setting.
The other system I wanted to mention in brief is the Rio Tinto. The Rio Tinto in southwestern Spain is distinguished by the fact that it has very high iron concentrations, and it’s a very low-pH (very acidic) environment. We were interested in that system initially in terms of the eukaryotic diversity. And if you look at eukaryotic diversity in that system, once again, you run into the same thing, namely that you find nearly all the possible eukaryotes you can imagine in the Rio Tinto.
What we’ve learned in Rio Tinto is that protists (single-celled eukaryotes) can operate at very low pHs. What strikes me about the system is that many of the protists that we see in the Rio Tinto are very closely related to organisms that are in culture collections today – organisms that don’t live in acidic conditions. What that effectively means is that adapting to an acidic environment like the Rio Tinto doesn’t take much time in an evolutionary time frame context.
What about the archaea and the bacteria in the Rio Tinto? One finds limited archaeal diversity in the Rio Tinto. And there are only half a dozen or so different bacteria that dominate the system. Is that an adequate description of the diversity of the Rio Tinto? We are not sure.
When you do molecular surveys, you’re constrained by how much you sample. How much you sample is constrained by how much money you have. So if you’re thinking about doing a sequence analysis and each sequence costs you a couple of dollars, if you want to count 10,000 samples, you better have a very well-funded grant.
We have started to explore an alternative methodology to deal with this problem. It’s called “serial analysis of ribosomal RNA sequence tags” – or SARST-V6 for short. The idea is to try to get a measurement not only of diversity in a natural environment, but also to get estimates of how many there are of each different kind of organism in a given environment. So that means we’re going to somehow sample thousands and thousands of sequences.
The technique that we settled on involves the idea of stringing together short stretches of ribosomal RNA from different organisms. We focus on a region of rRNA called V6, roughly 60 or 70 nucleotides in length, which is highly variable from one organism to the next. It’s flanked by some green-highlighted sequence elements. This V6 region is and it’s flanked on either side by a very conserved sequence, a part of the ribosomal RNA that is pretty much the same for all organisms. Our notion was that, if we could extract the short V6 sequence from ribosomal RNAs, and randomly string them together like beads on a string and then determine the structure of those short sequences, each short sequence would be a proxy for the occurrence of an organism in the environment.
The advantage of this technique is that now we can get out information about 10 to 20 different organisms for what it previously cost us to get information about only 1. It can’t give you information about deep phylogenetic relationships. But it’s useful for monitoring changes in population structure, or for measuring the relative abundance of organisms in a population.
When we tested the system initially in Guaymas, we got essentially the same kinds of distribution patterns, in this moderately complex environment, as we had with more traditional techniques. But while older techniques found Rio Tinto dominated by just 3 or 4 kinds of organisms, the SARST-V6 analysis shows these 3 or 4 kinds or organisms to account for just over 80 percent of the total population. When we go to the next 10 percent, we see a much larger array of organisms. We start to pick up many organisms that were previously invisible. And if we then include organisms that show up only a handful of times, we see still more organisms, which we’d never see by traditional sequencing.
So you see that the Rio Tinto is not made up of just a few organisms, but rather is made up of a moderately complex environment. That means that we shouldn’t just be paying attention to the dominant organisms at Rio Tinto, but we also have to pay attention to the minor players. Based on this new analysis, we now think that Rio Tinto is not 3 or 4 or 5 organisms, but it’s going to be on the order of hundreds of different kinds of organisms.
