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Microbe probe

Studying bacterial genomes

In 1956 a new microbe was discovered in a can of spoiled ground beef thought to have been sterilized by radiation. The bacterium was named Deinococcus radiodurans because it can survive doses of radiation thousands of times higher than would kill most organisms, including humans. The Department of Energy is interested in studying D. radiodurans because of its efficiency in repairing its own radiation damage and because it can reduce, or add electrons to, uranium, iron, chromium, and technetium. Thus, the bacterium might be a strong candidate for remediating mixed wastes—combinations of radioactive materials and toxic metals—found at DOE sites. For example, it could be used to convert uranium in storage ponds from a soluble to an insoluble form so that it sinks into the sediments instead of remaining in water that might flow off-site.

ORNL researchers are interested in knowing more about the bacterium's genes that repair radiation-induced damage to its DNA. Knowing how these DNA repair genes work together in a network could help scientists better understand how living organisms evolved and how microbes that may have lived on Mars or other extraterrestrial sites tolerated extreme radiation environments. This knowledge may lead to gene therapy in which DNA repair genes are inserted into the body to prevent radiation-induced cancer or to treat the disease. DNA repair genes discovered in D. radiodurans could be transferred to other bacteria that reduce toxic metals, to make them better able to survive radiation as they treat mixed wastes.

Thanks to advanced technologies such as microarrays and mass spectrometry and internal funding from the Laboratory Directed Research and Development Program, six ORNL researchers are improving the understanding of D. radiodurans. One of their goals is to identify which genes in D. radiodurans are expressed during exposure to high levels of radiation, resulting in the production of DNA repair proteins. Another goal is to discover the regulatory genes that control other genes involved in radiation resistance. The researchers are Jizhong Zhou, Bob Burlage, and postdoctoral scientists Dorothea Thompson and Alex Beliaev, all in the Environmental Sciences Division (ESD); Bob Hettich of the Chemical and Analytical Sciences Division, and Randy Hobbs of the Research Reactors Division.

To identify the D. radiodurans genes involved in DNA repair, Zhou and his ESD colleagues use microarrays, also known as gene chips. (See Complex Biological Systems in Mice.) They will place the entire complement of genes from D. radiodurans on the same gene chip and then use the chip to identify differences in global gene expression between D. radiodurans cells exposed to high radiation and those not so exposed. (The expressed genes produce messenger RNAs that can be detected.) Genes expressed only in the irradiated bacteria are likely to play specific roles in DNA repair.

The ORNL researchers will then develop genetic vectors for generating deletion mutants—bacteria from which key regulatory genes involved in radiation resistance are removed. These regulatory genes code for proteins that may activate other genes involved in DNA repair. If the mutant bacteria die when exposed to high radiation doses, then the results of this experiment suggest that the deleted genes play a key role in radiation resistance.

To understand the cellular response of the D. radiodurans bacteria to radiation, it is necessary to identify protein expression profiles and protein-DNA interactions. Zhou and his colleagues will culture the bacteria under both non-irradiated and irradiated conditions. The bacteria cells then will be lysed and the proteins will be extracted, purified, and finally characterized by Hettich, using high-resolution electrospray mass spectrometry.

"The capability of this technique for accurate measurement of the protein masses, as well as identification of sequence tags (short amino-acid sections from the protein), provides unparalleled information for unambiguous protein identification," Hettich says. Such information will be compared to that obtained from more conventional two-dimensional gel electrophoresis. The data should provide insights about how the microbe's proteome—its total protein profile—is altered in response to DNA damage, providing important details about gene activity.

"Knowing when and where a gene is expressed often provides a strong clue to its biological function," Zhou says. "Gene expression patterns and protein profiles in a cell can provide detailed information about its state." The researchers' goal is to identify pathways of interactions between DNA and proteins that result in DNA repair of radiation damage.

Another group of researchers interested in D. radiodurans is the Computational Biology Section of ORNL's Life Sciences Division. Using gene-recognition algorithms and other tools, Frank Larimer and his colleagues analyze completed DNA sequences of microbes and compare the identity and order of these DNA bases with known sequences in databases. They identify the known genes contained in these microbial genome sequences and predict the amino-acid sequences, molecular sizes, and possible functions of the proteins these genes encode. They also predict the makeup and functions of unknown genes and proteins.

"We have studied the completed sequence of D. radiodurans and can recognize many of its DNA repair genes," Larimer says. "We inferred the function of their proteins based on their degree of similarity with proteins in the database associated with repair genes.

"The majority view is that there is a 'eureka' DNA repair gene to explain why D. radiodurans is so resistant to high levels of radiation. I take the minority view that, although the bacterium's DNA repair genes look like the repair genes in other organisms, D. radiodurans does the repair job more efficiently. It is extremely adept at aligning and recombining its broken chromosome parts in the proper order."

D. radiodurans is one of the millions of microbes that have been evolving on the earth over the past 3.8 billion years. Many of the 1% of the microbes known to humans survive and thrive in extremes of radiation, heat, cold, pressure, salinity, and acidity, often where no other life forms could exist. DOE is interested in identifying and harnessing the talents of some of these microbes for cleaning up hazardous wastes, producing energy (e.g., methane), and sequestering carbon.

Since its establishment in 1994, DOE's Microbial Genome Program has focused on determining the sequences of the genomes of selected bacteria and other microbes that do not cause disease. The ORNL group provides computational and other bioinformatic analysis of microbial sequences obtained by DOE's Joint Genome Institute (Lawrence Berkeley, Lawrence Livermore, and Los Alamos national laboratories) at their production facility in Walnut Creek, California. Larimer and his colleagues "annotate" these microbial DNA sequences and others provided by academic groups. That is, they add to the database "biological footnotes" about the genes, the coding and noncoding regions of the genes, and the possible structure and function of the proteins encoded by individual genes. For each microbe, the researchers translate roughly 2 billion DNA bases into meaningful information.

Using the IBM RS/6000 SP supercomputer at DOE's Center for Computational Sciences at ORNL, the group runs GrailEXP, the version of the ORNL-developed Gene Recognition and Analysis Internet Link written for parallel supercomputers, along with other gene modeling programs to determine the correct DNA sequences in the microbial genes that code for proteins. "Ninety percent of bacterial DNA encodes protein compared with less than 2% for humans," Larimer says. "Microbes are very efficient this way. So we try to distinguish between what portions of the bacterial sequence are genes and what is a statistical anomaly."

The typical bacterium has 2000 genes (each microbial gene has about 900 bases). The ORNL group tries to find gene signatures in bacteria—unique combinations of genes that indicate each bacterium's identity.

"What is startling is that we can assign functions for 50% of the genes in a microbial gene, which might be 1000 genes. The other half of the genes have no known function. Half of these genes are unique—we've never seen them before. There is a lot of genetic biodiversity in microbes. Microbes acquire sets of genes from each other and throw away large blocks of genes they don't need as they evolve."

To date, 50 microbial genes have been completely sequenced. Larimer's group has been annotating them and working with 200 ongoing microbe sequencing projects. DOE's goal is to sequence 250 microbes that could be useful for its energy and environmental missions.

In addition to D. radiodurans, some of the bacteria being analyzed are particularly fascinating to Larimer. One is Prochlorococcus marinus, a blue-green marine alga that is the most abundant organism on earth in number and mass. These ocean algae, whose individual cells are each less than 1 micron in diameter, together fix more carbon dioxide than all of the terrestrial biosphere. Prochlorococcus marinus is found 100 meters deep in the ocean yet it absorbs sufficient blue-green photons penetrating to that depth to get enough energy to fix carbon.

Another interesting microbe that fixes carbon dioxide is Nitrosomonas europa, which gets its energy not from sunlight but from oxidizing ammonia. Using ammonia fertilizer, it is responsible for putting nitrogen into the soil, making it fertile for plants.

Zhou is fascinated by gene-chip and mass spectrometry studies of the metal-reducing bacterium Shewanella oneidensis. "We compared gene expression patterns in Escherichia coli bacteria and a mutant of Shewanella oneidensis that we generated," Zhou says. "We found that the functions of about 75% of their genes are similar. But there are differences. In E. coli one gene activates nitrate reduction, but the counterpart gene in mutant Shewanella oneidensi represses nitrate reduction.

"Using bioinformatics, scientists often assign gene functions based on the similarity of sequences of two different bacteria. This approach may not correctly define all gene functions. To get the complete picture, bioinformatics must be supplemented by experimental approaches that analyze expression in microbes, using microarrays and mass spectrometry."



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