Scientists Report First Sequencing Of Environmental Genome

The researchers took a simple community of microbes from a pink slick on the floor of an abandoned mine, ground them up, and shotgun sequenced the lot. As they put the pieces of DNA back together, the snippets fell easily into five distinct genomes, four of them unknown until now.

"This is the first recovery of a genome from an environmental sample," said Jillian F. Banfield, professor of earth and planetary science and of environmental science, policy and management at UC Berkeley. "This ushers in a whole new way of exploring and understanding our environment, allowing us to determine how organisms work as individuals and together, and how they contribute to geochemical processes."

Banfield and graduate student Gene W. Tyson from UC Berkeley's Department of Environmental Science, Policy and Management, with colleagues from UC Berkeley and the U.S. Department of Energy's Joint Genome Institute (JGI) in Walnut Creek, Calif., report their feat this week in the Advance Online Publication of the journal Nature.

Banfield and her students, post docs and colleagues are primarily interested in how the microbes, obtained from the Richmond Mine in Iron Mountain, Calif., one of the largest Superfund sites in the country, interact with minerals to produce acid mine drainage.

"Acid mine drainage is one of the most pressing long-term environmental problems worldwide, and it's caused by microbial processes," Banfield said. "This study has dramatically improved our understanding of the microorganisms involved and has opened the way for development of much more highly refined models of acid mine drainage systems."

"If we understand the organisms and how they cause this environmental problem, we can try to do something about it in the long run," Tyson added.

"This represents an important example of how the production sequencing capacity developed by the Department of Energy at the JGI for the human genome program can provide fundamental insights into vital environmental problems," said JGI Director Eddy Rubin.

Understanding the biofilm ecosystem also may be relevant to the search for life on Mars, since it's conceivable that the iron and sulfur-rich surface of Mars could harbor microbes that eat iron, similar to those in iron and sulfur-rich pyrite mines like the Richmond Mine.

For the past nine years, Banfield has been studying a pink microbial biofilm that sits like scum on the surface of green pools of water, as acidic as battery acid, in the dark depths of the Richmond Mine, located nine miles northwest of Redding. Her goal is to understand how the extremophiles - microbes that live in extreme environments - live together and generate the acid drainage that makes such mines toxic hazards. The green runoff from the mine, captured and treated by the Environmental Protection Agency, is not only acidic, but also contains high levels of toxic metals - zinc, iron, copper and arsenic - and is a piping 108 degrees Fahrenheit.

In this low-light, low-oxygen, high-acid and toxic environment about 1,400 feet into the mountain, the microbes thrive. They fix carbon and nitrogen from the carbon dioxide and nitrogen in the air, eat iron by oxidizing it with oxygen, and in the process dissolve the iron pyrite (iron sulfide, also known as fool's gold) to create sulfuric acid.

Previously, researchers have studied microbial communities, such as those in hot springs or in the ocean, either by isolating individual organisms or strains, culturing them and sequencing the cultured population; or by plucking bits and pieces of genes from the various members of the community.

A big problem is that only about one in 100 microbes can be cultured sufficiently to extract its genome. Even if a microbial genome is known, however, this still doesn't tell researchers how it interacts with other microbes in its environment.

Banfield and other researchers have been looking at a more daring approach - sequencing the whole community at once, a technique Banfield prefers to call "community genomics." That's like surveying the species in the African veldt by grinding up lions, zebras, elephants and an unknown number of other animals, cutting the genes into tiny pieces, and trying to sort them into distinct genomes.

But it works, Banfield said, "at least with the small number of distinct organisms in this community."

"The magic of the whole thing is that, because of speciation, these organisms are different enough that their genomes are easy to tell apart," Banfield said. The technique would allow researchers to sequence the genomes of microbes that cannot be raised in isolated cultures.

Banfield's group and the group at the JGI reassembled the genomes, each containing about 2,000 genes, using different software programs to arrive at composite genomes. Two of the draft genomes - for a Leptospirillum group II bacterium and a Ferroplasma type II microbe from the ancient group known as Archaea - are now about 97 percent complete, with a few gaps. The genome of one of the six microbes in the community, Ferroplasma acidarmanus (a type I Ferroplasma), had been sequenced earlier by JGI and Banfield's group and was a good control during the sequencing and assembly process.

The other genomes were highly fragmented, but identifiable as microbes from Leptospirillum group III, Ferroplasma type I and a G-plasma microbe.

"Despite the highly fragmented nature of three of the genomes, we had enough coverage and many, many genes to get an idea of what the organisms do in the environment," Tyson said.

The genome of Leptospirillum group II is the first sequenced from the phylum Nitrospira, an important branch of the tree of life containing nitrate and nitrogen cycling bacteria, Banfield noted.

Once Banfield's group had the five new genomes, they compared the genes in each with a database of known genes to identify their functions. For the two microbes with the most complete draft genomes, they were able to reconstruct nearly the entire metabolic cycle.

Already they have determined how the microbes share tasks in the isolated microbial community of the mine. The Leptospirillum group II bacteria fix carbon and produce the biofilm that protects them and keeps them afloat, while a minor member of the community, Leptospirillum group III bacteria, fix both carbon and nitrogen. The iron is probably munched by the all of the biofilm members, including Ferroplasma microbes.

Interestingly, the Ferroplasma type II microbes in the biofilm apparently are a mix of many different strains, but the genome reconstruction shows that they all arose from three distinct ancestral strains. Random sex among the microbes for millions of years has combined and recombined the genomes of the three strains so that the population today consists of strains that mix and match genes from separate ancestors.

"This type of rampant exchange of genetic material has never been documented in Archaea or in natural samples before," Banfield said. "This recombination may be a strategy for maintaining optimization in the event of perturbations in the environment. The observation helps us understand the factors that shape evolution and drive development of new species."

Banfield said that the community genome technique worked in this situation because the dominant microbes were represented mostly by closely related strains. In more complex environments with more organisms, it may not be so easy.

"However, even in more complex environments it should be possible to extend the random shotgun sequencing approach to recover genomes of uncultivated strains and species," the authors wrote. "These data can then be used to explore the nature of the community metabolic network, to find conditions for cultivating previously uncultivated organisms, to monitor community structure over time, and to construct DNA microarrays to monitor global community gene expression patterns."

While the current study provides a wealth of information about the five microbes, it's only the beginning for the team. With the genes in hand, they plan to create microarrays - so-called gene chips - to determine how gene expression and protein production change with changing conditions in the microbial community. For example, as the acidity or temperature changes, how do the organisms react? How do they change their carbon or nitrogen fixation, or their scavenging of iron from the acidic groundwater?

"The next step in this project is to get whole community genome arrays up and running to look at these organisms and how they respond to certain conditions," Tyson said.

Other authors include Paul Richardson and Victor Solovyev of the Joint Genome Institute; Daniel Rokhsar and graduate student Jarrod Chapman of both JGI and UC Berkeley's Department of Physics; and scientist Philip Hugenholtz and post-doctoral researchers Eric E. Allen and Rachna J. Ram of UC Berkeley's Department of Environmental Science, Policy and Management.

This research was funded by the US Department of Energy's Office of Science and the National Science Foundation's Biocomplexity Program.