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Creatures from the Black Lagoon:
Lessons in the Diversity and
Evolution of Eukaryotes (4 of 5)

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III. Uncultivated Microbial Eukaryotes

Characteristics of eukaryotes that live without oxygen
Most known anaerobic protists are pathogens. One could argue that the only reason we know about the ones we do is becuase they affect us — they cause disease. Anaerobic protists have evolved multiple times (which is termed "polyphyletic") but since the most basal eukaryotes are anaerobes, we believe the original eukaryotic cell evolved in an anoxic world. How might we look for eukaryotes still alive today, which might be more genetically closer to those ancestral types, than to more recently evolved groups like us?

Identifying protists by microscopy
One way we could do this would be by microscopy. Antoine van Leeuwenhoek, a lay person (not a card-carrying scientist), really began the study of microbiology in the 17th century with the use of one of the first microscopes. van Leewenhoek described the first microorganisms — he called them "animalcules." People still identify microbes based solely on microscopic descriptions today. These descriptions can read like field guides. Though our microscopes have gotten a lot more sophisticated since van Leewenhoek (e.g., electron microscopes), are there other ways to find these relict eukaryotes?

Identifying protists by cultivation
Well, we could grow them. We know Louis Pasteur for a lot of work with microbiology, such as germ theory and pasteurization. But he was also the first to describe organisms which were able to grow in sealed flasks without "air." This was truly a first, as people at that time believed that all organisms needed air, meaning oxygen, to grow.

But we encounter problems when we rely only on visualizing or growing organisms in order to identify them. First and foremost, it can be really hard! Think if we had to grow every animal in the zoo or every plant in the forest in order to know they were there. Plus, we don't do a really good job at growing most microbes. It's estimated that only about 1% of all microbes in the natural world have been grown. Fortunately, there's a better way to identify organisms — not by how they look or how they grow, but by their DNA.

Identifying protists by their genotype
DNA. We hear a lot about DNA, the genetic material of all organisms is always in the news. You've probably heard about DNA fingerprinting to identify suspects in crimes, or prove paternity. Well, this method of DNA fingerprinting has been used extensively in the past 15 years or so to identify uncultivated bacteria and archaea. And, using these new molecular techniques, we've discovered that the microbial diversity of bacteria and archaea has been grossly underestimated. Our study, and the rest of the work I'll present today is the first to look for uncultivated microbial eukaryotes, or protists, by their DNA fingerprints. How do we do this?

A tablespoon of mud We start with about a tablespoon of mud — remember that this can contain several billion microbes. This is mud from Berkeley Aquatic Park. Then we extract the DNA from the mud. By the way, the black color of the mud is a good indicator that there is no oxygen at that layer. Reduced iron complexed with sulfur produces something called pyrite — a dark black precipitate.

Xeroxing DNA
The polymerase chain reaction, or PCR, is basically a way to xerox DNA so that we have enough to work with in the lab. Many microbes are rare and we need to amplify their DNA signals to be able to find them. In our work we're xeroxing a specific gene — the gene for rRNA. Remember this is what Carl Woese used to relate different microbes to each other and construct a tree of life.

Aligning genes
Once we get the DNA sequence, we line up the sequences and compare them to known organisms. The rows correspond to an rRNA sequence from a particular organism; the column relates the exact nucleotide (A,T,C,G) at a specific position in the sequence of the gene. Note the conserved/variable regions.

Constructing a phylogenetic tree
Then, from this alignment of DNA rRNA sequences, we use computer models to simulate the evolution of sequences. We infer that the evolution of the sequences corresponds to the evolution of the organisms. In this way, we are able to determine evolutionary relationships among the new uncultivated microbes. Unlike Linneaus, Darwin or Haeckel, we organize geneologies based on genotype, not phenotype. This also gives us a picture of evolution. Note the organisms in blue — those we only know from environmental sequences or fingerprints. Also note how we can compare where they fall on the tree to known organisms. OK, let's talk more about our study of creatures from the Black Lagoon.