Capturing gene expression in a natural setting

Summer 2008

PROBLEM

We know that ocean microbes act like tiny biosensors, responding quickly to changes in the environment in ways that help us clean up chemical spills or treat raw sewage. But we know little about how they do it, including the genes and metabolic pathways involved. Metagenomics—the study of genetic material of microbial communities collected from natural environments—is a burgeoning field of genetic research particularly useful for the study of ocean microbes, because they’re difficult to culture in a lab. By focusing on the microbe’s RNA transcript profile, Professor Ed DeLong of MIT’s Department of Civil and Environmental Engineering, one of the pioneers of metagenomics, now brings us a step closer to identifying which genes are active under certain conditions.

RNA profile provides a true picture of a microbe’s gene expression at home at the time of capture, but that picture can alter very quickly. Within even a few minutes of its removal from its natural oceanic home, the marine microbe’s gene expression may adjust to changes in light and temperature. This presents a major hurdle in the collection of samples of microbial RNA. How can you collect and filter enough seawater to get sufficient numbers of the single-celled organisms before they begin to significantly change their genetic expression?

APPROACH

In a paper published in the March 11 issue of the Proceedings of the National Academy of Sciences, DeLong, Professor Sallie Chisholm, Jorge Frias-Lopez, Ph.D. student Yanmei Shi, and co-authors describe a method of amplifying the RNA extracted from the small sample of microbes that can be collected in a short time. The method involves modifying a eukaryotic RNA amplification technique, and generating complementary DNA from amplified RNA.

FINDINGS

The research team tested the fidelity of the technique by comparing levels of gene expression from amplified RNA with those of unamplified RNA extracted from Prochlorococcus cultures. (Prochlorococcus is the most abundant photosynthetic microorganism in the ocean). The gene expression profiles compared favorably and the amplified RNA revealed the same physiological response to phosphate starvation as did the unamplified RNA: by up-regulating the same four genes.

The team field-tested the methodology by taking samples of seawater in the North Pacific. In about 10 minutes, they gathered and filtered about 1 liter of seawater to get microbial biomasses, from which about 100 nanograms of total RNA were extracted. They were able to amplify that RNA 1,000 times. About 50 percent of the genes in this sample had not been documented in previous databases of protein-coding genes. Highly expressed genes included those associated with known ecologically important metabolic pathways (including photosynthesis, carbon fixation and nitrogen acquisition). The sample also contained a number of highly expressed “hypothetical” genes—sequences that look like they might encode for proteins with unknown functions.

IMPACT

This methodology promises to yield much new information to advance our understanding of, and ability to predict, microbial metabolic responses in natural or engineered environments. DeLong and colleagues are now using the technique to look at microbial gene expression in response to an influx of carbon, the addition of nutrient-rich deep seawater to surface waters, and over time. This study could lead to identification of the metabolic pathways involved in marine microbial reactions to certain nutrients.

Delong was elected to the National Academy of Sciences this spring, and received major awards from the European Geosciences Union and the American Society of Microbiology.

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