Collaboration at a Superfund Site
ORNL scientists play key roles in the first comprehensive study of microbial gene expression at an acidic mine.
Using mass spectrometers and bioinformatics tools, Oak Ridge researchers made key contributions to the first comprehensive study of gene expression in a community of microbes thriving in an unusually harsh acidic environment. Their work helped their collaborators understand how these microbes survive as a community in a Superfund site at Iron Mountain, California.
In a paper published May 5, 2005, in the online Science Express, scientists from Lawrence Livermore and Oak Ridge national laboratories, the University of California, Berkeley, and Xavier University in New Orleans identified more than 2,000 proteins produced by five key microbial species in the community. More than 500 of the proteins appear to be unique to the community, and many do not resemble known proteins.
The proteins were identified largely by ORNL chemist Bob Hettich and Nathan VerBerkmoes, a postdoctoral researcher, both in the Chemical Sciences Division. Using "shotgun" mass spectrometry, they determined the molecular masses and fragmentation patterns of proteolytic peptides from these proteins To identify the peptides from the experimental data, they turned back to the genomes. These DNA sequences of microbial genes in this natural community were identified in 2004 by the Department of Energy's Joint Genome Institute in California and ORNL researchers. The sequences specify the order in which chains of amino acids are linked together to make proteins.
The genomes allowed Manesh Shah of ORNL's Life Sciences Division and other researchers to predict the proteins in each member of the biofilm community. Shah, for example, used computational tools to search the microbial genome database. He then disseminated appropriate data to help the researchers predict the protein fragments that would result if the proteins were chopped into pieces. Correlating the experimental results with the predicted data allowed the researchers to reconstruct the complete proteins and then associate each protein with a particular organism.
"This first large-scale, proteomics-level examination of a natural microbial community from the environment demonstrates the best approach for conducting research in systems biology," Hettich says of the DOE-funded work. "We assembled a strong multidisciplinary research team with critical expertise in each area of systems biology." (See systems biology issue of ORNL Review, Vol. 37, No. 3, 2004.)
Jill Banfield, professor of earth and planetary science and of environmental science, policy, and management at UC Berkeley, leads a research team that has conducted a nine-year study of the Iron Mountain microbial community, which consists of bacteria and archaea. The study was the first microbial community characterized at the genetic level. Banfield's team extracted samples of the pink biofilm floating on the water at the abandoned iron mine and prepared them for mass spectrometry analyses.
Michael Thelen, a protein biochemist at Lawrence Livermore, helped unravel the functions of some extracellular proteins in the microbial community. He determined that many unfamiliar proteins are enzymes whose function is to maintain the correct structure of other proteins exposed to sulfuric acid and toxic heavy metals.
Thelen also found that some microbes specialize in fixing nitrogen for the community. One bacterial member of the community, Leptospirillum group II, makes a cytochrome protein in abundance, which may capture an electron as the first step in oxidizing iron, a source of energy that allows the intake of airborne nutrients—carbon and nitrogen. The iron was leached out of iron sulfide rock.
"We also found that a different bacterium, Leptospirillum group III, apparently makes more of the polysaccharide that is needed as a matrix or housing material for the biofilm community," Thelen says. "These organisms evolved to adapt to their specific environment. They come up with genes distinct enough from those of other organisms to enable them to survive in that particular niche."
Raymond Orbach, director of DOE's Office of Science, says, "Now scientists can investigate not only the 'community genome' but also the resulting 'community proteome' for enzymes and pathways that can help clean up some of the worst environmental sites in the nation."
Web site provided by Oak Ridge National Laboratory's Communications and External Relations