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Pioneering Study Used Mass Spec to Analyze Proteome of Entire Microorganism Community

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University of California Berkeley researchers have used mass spectrometry and bioinformatics techniques to study the proteome of an entire community of microorganisms for the first time.

The researchers, led by Rachna Ram, a postdoctoral scientist, and Jillian Banfield, a professor in the department of environmental science, policy and management at UC Berkeley, said they decided to study the proteins within all the organisms collected from an acid mine drainage environment, rather than study the proteomes of individual organisms, because data from a multi-organism community collected from its natural environment is more representative of what is happening in nature than individual organisms cultured in laboratories.

The research, which is believed to be the first of its kind, potentially adds broader significance to the proteomic community because it paves the road for the proteomes of other types of communities to be studied as well.

"It makes sense, because in the microbial ecology world you should be thinking about how microorganisms interact with each other in mixed communities in nature," said Ram, the lead author of a paper describing the study, which appears in the June 24 issue of Science. "If you study each individual organism, you might be missing some of the picture by taking them out of the context of where they were in nature."

The research stemmed from a genomic study also done by Banfield's research group on the same acid mine drainage community, located at a Superfund site outside Redding in northern California. For that study, published in March 2004 in Nature, members of Banfield's research group collected the surface layer, or biofilm, from pools of acid mine drainage, isolated DNA from the samples, shotgun-sequenced the DNA, and used knowledge of the relative abundances of the relatively few organisms within the community to reconstruct the genome of each organism within the community.


"It makes sense, because in the microbial ecology world you should be thinking about how microorganisms interact with each other in mixed communities in nature. If you study each individual organism, you might be missing some of the picture by taking them out of the context of where they were in nature."

That study "was the first time anyone had characterized the genomic content of a natural microbial community," said Banfield. "We then wanted to turn to proteomics in order to understand [what molecules] are doing what, when, and how."

By matching protein sequences with genomic sequences from the 2004 study, Ram and her colleagues were able to not only look at proteins within the whole community, but also to separate out which proteins came from which organisms.

Ram and her colleagues detected 2,033 proteins from the five most abundant species in the acid mine biofilm. Forty-eight percent of the predicted proteins came from the dominant biofilm organism, Leptospirillum group II. Proteins involved in protein refolding and response to oxidative stress appeared to be highly expressed, suggesting that the organisms' response to biomolecular damage is a key challenge for survival.

"One of the things we were able to figure out was which proteins in the organisms might be exposed to the acidic environment. Those proteins could have new properties," Ram said. "Another one of the main things was that novel proteins seemed to play a really important role. We found a lot of proteins that don't look like any other proteins identified and characterized in any other organism."

In terms of practical applications of her study, Ram said that if researchers can understand how microorganisms, including Leptospirillum, oxidize iron, they might be able to speed up the oxidation process, thereby decreasing the amount of time it will take for the acid mine to become non-acidic or less acidic.

The acidity of the mine comes from the oxidation of a sediment called pyrate. When pyrate oxidizes, it releases ferric iron. Without the presence of microorganisms, the ferric iron would deplete quite quickly, but microorganisms regenerate the ferric iron, thus playing a role in creating the low pH environment.

The acidic drainage and toxic metals from the mine have created environmental problems in the past, including fish kills in the Sacramento river. Now the acid mine is managed by the US Environmental Protection Agency, which takes care of eliminating acidity and toxic metals from the mine's drainage before it reaches the waterways.

At the current rate of pyrate dissolution, it will take 2000 years before the pyrate mineral runs out and the mine is able to become less acidic, Ram said. If scientists can find out what conditions in nature could hasten iron oxidation, they might be able to mimic those conditions and speed up the dissolution of pyrate, she speculated.

"By understanding how the community is optimized, we could accelerate the problem of producing acid mine drainage," said Banfield. "Another application if we could optimize the process is we could actually recover the metals, such as copper and zinc, in a low-cost, effective, environmentally friendly way."

Another application of the current research may involve using some of the novel, acid-stable proteins for industrial processes.

"People are very interested in novel enzymes, and these [acid-stable] enzymes may have some value," said Banfield.

Ram says that the next step in pursuing this field of research will involve looking at different biofilms within the same environment and trying to correlate changes in expression with changes in the condition the community is found in, given that different biofilms may vary a bit in temperature or pH.

In addition, Ram plans to look at how protein expression in a cultured microorganism isolate is different from protein expression within a natural community.

"We're trying to compare the proteomics of different communities, and to tell how community affects the behavior of an organism," Ram said.

Ram said she has heard of other research groups that want to investigate the proteomes of other types of communities, but so far the only other group that has published a community-level molecular study is Craig Venter's group, which last year published a study on the genomics of a sample from the Sargasso Sea. However, that study was more geared toward looking at how diverse things are in the natural environment, Ram said, rather than characterizing a simple community's genome and proteome, and looking at the effect of a community on the behavior of organisms.

— Tien-Shun Lee ([email protected])

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