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Release: Sept. 24, 2001

UI microbiology researchers receive grants to study bacterial genes involved in biodegradation

IOWA CITY, Iowa -- Two University of Iowa researchers will use three grants from the U.S. Department of Defense (DOD) and the U.S. Department of Energy (DOE) to study bacteria that can generate a natural fuel, mop up a greenhouse gas and biodegrade toxic waste.

Rebecca Parales, Ph.D., UI research scientist in microbiology, has received a four-year, $583,951 grant from the DOD to study the ability of bacteria to biodegrade toxic compounds.

Working in collaboration with Jim C. Spain, Ph.D., at Air Force Research Laboratory, Tyndall Air Force Base, Fla., Parales will investigate how certain bacteria can biodegrade toxic compounds called dinitrotoluenes (DNTs), which are produced when the explosive, trinitrotoluene (TNT), is manufactured.

These compounds are completely man-made and did not exist until about 100 years ago. The bacteria that use these compounds as energy sources did not start to evolve until the compounds were introduced into the environment. To date, these bacteria have only been found at sites that are polluted with DNTs and TNT.

"We know of organisms that can grow on other aromatic compounds such as benzene," Parales said. "However, these nitroaromatic degraders seem to be quite specialized. I think they are evolving now.

"The organisms we are isolating now are more efficient at degrading DNTs than those we isolated 10 years ago," Parales added.

One goal of Parales's grant is to fully characterize these bacteria and learn which genes and biochemical pathways are involved in the breakdown of DNTs. A better understanding of how these bacteria perform their biodegradation could suggest ways to improve the efficiency of these reactions.

A second goal is to try to create new strains of bacteria, which can also degrade TNT to a non-toxic form even as they use DNTs as an energy source. So far, the researchers have not found any naturally occurring bacteria that can degrade TNT.

The researchers will use a process, known as directed evolution, to randomly shuffle the DNA encoding a bacterial enzyme that partially degrades DNTs. This approach will create a library of slightly different proteins, some of which may be able to partially degrade TNT.

Caroline Harwood, Ph.D., UI professor of microbiology, has received a three-year, $315,000 grant from the DOD to study bacterial genes involved in biodegradation of aromatic molecules. However, rather than investigating bacteria that have evolved in polluted environments and specifically degrade toxic compounds, Harwood's research focuses on Rhodopseudomonas palustris (R. palustris), a bacterium that is commonly found in many environments. This bacterium, whose genome has been fully sequenced, can biodegrade benzene rings in the absence of oxygen.

"Knowing the genome sequence, we can now spot candidate genes that are likely to be involved in related pathways," Harwood said.

Harwood and her colleagues believe that studying biochemical pathways in R. palustris will provide useful information about similar pathways in bacteria that survive in polluted environments but are much more difficult to grow and study in the laboratory.

"R. palustris provides a good model for these interesting but hard-to-study bacteria," Harwood said. "What we learn about R. palustris may suggest other routes for using biology to remediate toxins."

The UI researchers will use a technology known as microarray analysis to confirm which genes are part of which biodegradation pathways.

In addition to its ability to degrade benzene, R. palustris has several other interesting features, which Harwood also is studying. Using a three-year, $800,000 grant from the DOE, Harwood and her team will investigate the bacterial genes involved in carbon dioxide and nitrogen fixation.

Plants and bacteria, including R. palustris, remove carbon dioxide from the atmosphere in a process known as carbon dioxide fixation.

"Carbon dioxide is a major contributor to global warming," said Harwood. "The Department of Energy is interested in better understanding any living process which removes carbon dioxide from the atmosphere."

In addition to carbon dioxide fixation, R. palustris can also convert atmospheric nitrogen into ammonia, a critical biological building block. Only bacteria can perform this transformation, known as nitrogen fixation, and introduce ammonia into the Earth's nitrogen cycle.

The enzymatic reaction, which converts nitrogen to ammonia, also produces hydrogen. The hydrogen production is actually a biological drawback for the bacteria as it makes the reaction very

energy intensive. However, it could be considered an advantage for humans because hydrogen can be used as a natural fuel.

The research team plans to identify genes that are important for these processes. Although the genome sequence is known, up to 40 percent of the genes code for proteins with unknown functions.

"It is very likely that some of these proteins are important for efficient carbon dioxide fixation and nitrogen fixation with concomitant hydrogen production," Harwood said. "We want to identify all the genes that are involved in these processes."

The team also will generate genetically modified versions of R. palustris to pin down how the molecular players in these processes interact with one another.

"Once we understand these pathways, we will have a better idea of how we might improve them either through genetic manipulation or by using different growth conditions," said Harwood. "We want to make them the best at doing what we want them to do."

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