Some like it hot
Yellowstone’s hot springs show vivid yellows, oranges, reds, ochres, greens, and blue-greens. Many of these colors are pigments produced by the different species of bacteria that exist on and within the roughly inch-thick microbial mats that form in and near the springs’ runoff channels. Octopus Spring earns its name from its complex shape. It’s an alkaline spring, with a pH of 8.5 (the pH scale ranges from 1 to 14, with 7 being neutral: neither acidic nor alkaline). Octopus Spring also has a high content of dissolved silica and a relatively low sulfide content. Its geothermally heated source releases 95-degree C. water (203 degrees F.)—very near boiling at the high altitude of Yellowstone, in the Rocky Mountains. Octopus Spring has two effluent channels in which the water, flowing away from its source, gradually becomes cooler: 66 to 50 degrees C., or 151 to 122 degrees F. In those channels float the gaudy mats that Bryant and Ward have been studying, in areas where the effluent cools to below 72 degrees C., the highest temperature at which photosynthesis can occur.
Cross-section of a microbial mat
"looks a bit like lasagna."Credit David M. Ward
The only living inhabitants of Octopus Spring are its microbes: The place is simply too hot for higher organisms to survive. A microbial mat is a world of its own, consisting of many layers and interlacing zones that support a range of different microorganisms. “The mat looks a bit like lasagna when you cut into it,” Bryant says. “In the one that we’ve been working on, you can move one centimeter away from our study area and find a bacterium that looks physically the same as a neighboring type, yet is sufficiently different genetically to be considered a separate species.
“We’ve been looking mainly at the upper two millimeters—the green part of the mat and what’s just beneath it.” Cab. thermophilum grows in this surface area, along with cyanobacteria. (Cyanobacteria are an exceedingly common and abundant group of bacteria also known as blue-green algae. They exist in the oceans, where they play a key role in the marine nitrogen cycle, and in a wide range of freshwater habitats. Scientists believe their oxygen-releasing photosynthesizing made it possible for life to evolve on Earth.)
Don Bryant at Mushroom SpringCredit David M. Ward
In Octopus Spring, “The upper layer of the mat, exposed to light and the atmosphere, is where all of the photosynthesis takes place,” Bryant says. “Other microorganisms in the lower layers of the mat live under anoxic conditions and do not carry out photosynthesis."
“The mats are complex communities,” continues Bryant, “with many synergistic relationships between the various microbes.” Because of those complicated and often poorly understood relationships—as well as the hard-to-duplicate physical and chemical characteristics of the hot springs habitat—thermophilic bacteria can be difficult or even impossible to grow in the lab. “Cab. thermophilum simply refuses to grow on an agar plate,” Bryant says.
In the hot springs, Bryant and Ward and their graduate students and colleagues take samples from the mats using low-tech methods: A spatula works nicely for cutting out a portion of the mat, as does a coring tool. (Neither device causes lasting damage, as the microbes grow back quickly.) Once a sample is brought into the lab, the target layer is removed. The researchers then use a technique called metagenomics to explore the organisms living in that part of the mat, and to puzzle out their physiology, metabolism, and ecological relationships.
How little we know
In traditional microbiology and microbial genome sequencing, researchers isolate bacteria by growing them as pure cultures on media such as agar—the classic culture in a test tube or a petri dish—and then take DNA from the pure strain and sequence it. Metagenomics is based on a completely different approach: DNA is harvested from the vast numbers of bacteria in an environmental sample and then analyzed through DNA sequencing, almost as if it were part of the genome of a single organism. The technique, developed in the 1990s, involves extracting DNA and then subjecting it to “shotgun sequencing,” in which many relatively short DNA sequences are assembled into “consensus sequence fragments,” or “contigs,” through the use of powerful computational techniques. Says Bryant, “The sequence contigs can then be further analyzed to reveal information about the organisms living in that environment, and also to infer those organisms’ physiological and metabolic processes.”
In sorting through their Octopus Spring samples, Bryant and Ward focused on two genes: 16S ribosomal RNA, part of the machinery used by all living cells to manufacture proteins, and distinctive from species to species; and the gene coding for a protein called PscA, which, because it is essential for converting light energy into chemical energy, is an indicator of photosynthesis.
“Finding two distinctive genes among thousands of DNA fragments is not enough to justify naming a new organism,” Bryant says. “You need to prove that those genes came from the same genome, from the same organism”—this in a part of the mat where no less than six phototrophic bacteria have already been identified. It turns out that the genes for PscA and 16S ribosomal RNA are physically close together in the chromosomes of Cab. thermophilum , and the research team was lucky enough to isolate a single large DNA fragment that included both. The odds of making such a connection were not high.