Among the first to identify bacteria that break down nitroglycerin, the active component of dynamite, University of Wisconsin-Madison researchers now have identified two enzymes that enable bacteria to degrade both nitroglycerin and TNT, another explosive. The scientists have sequenced the genes that code for the two enzymes.
“Others have found bacteria that can degrade nitroglycerin, but this is the first time anyone has purified and characterized the enzymes that can take the initial step in breaking down TNT,” says bacteriologist Glenn Chambliss, the project”s leader. The team includes enzymologist Brian Fox, environmental engineer Dan Noguera, bacteriology graduate student David Blehert and bacteriologist Kyle Knoke.
The two enzymes, each produced by a different Pseudomonas species, are flavoproteins. “Flavoproteins are common to many soil-dwelling bacteria,” says Chambliss, who chairs the Department of Bacteriology in the College of Agricultural and Life Sciences. “As a group, these yellow enzymes appear to help bacteria protect themselves from natural chemicals in the soil. Many flavoproteins can degrade a range of different chemicals.”
The findings may lead to biologically based methods for cleaning up sites contaminated with toxic residues left from manufacturing explosives, according to Chambliss. He hopes to identify or engineer bacterial strains that can help clean up such sites.
There are an estimated 10,000 U.S. sites contaminated with explosives and related compounds. The materials include: TNT (trinitrotoluene); DNT (dinitrotoluene); nitroglycerin; and nitrocellulose, also known as smokeless gunpowder. TNT and DNT are particularly thorny environmental contaminants because they are toxic and break down extremely slowly.
“My real love is trying to figure out what makes bugs tick,” Chambliss says. But as a youth in East Texas, he first developed a love of the outdoors. When the opportunity came along to wed his two passions, the former Madison Audubon Society president became interested in finding bacteria that might help clean up polluted sites. Ultimately, he wants to learn how bacteria, which have been around for billions of years, developed the ability to degrade man-made chemicals that first appeared about 150 years ago.
To find bacteria that could “eat” dynamite, Chambliss began at the Badger Army Ammunition Plant located 30 miles northwest of Madison, Wis. The plant operated during World War II, the Korean War and the Vietnam War. It was once the world”s largest producer of smokeless gunpowder, a propellant used to fire artillery shells.
Chambliss and his coworkers went to sites once contaminated with nitroglycerin. They isolated and identified six bacteria that could survive at high nitroglycerin concentrations and degrade the compound.
Now they have characterized the enzymes from two of the bacteria, Pseudomonas putida and Pseudomonas fluorescens. Chambliss says that about half of the amino acids that make up the two enzymes are the same. Both enzymes can degrade nitroglycerin and TNT. However, one is five times as efficient as the other at degrading TNT. Chambliss notes that this single enzyme constitutes 15 percent of the cell”s protein, an unusually high percentage.
The more-efficient enzyme can follow two different pathways in degrading TNT, according to Chambliss. “One leads to toxic components that don”t decay further. The other pathway leads to a partial but more complete breakdown without toxic compounds.”
Chambliss is now experimenting to see if he can engineer the enzyme so it only works via the preferred pathway. He is also looking for other bacteria and enzymes that will complete the cleanup.
“The bugs have the potential to address soil contamination,” Chambliss says. “But we”re still at an early stage in understanding the processes involved. It”s going to take more research before we get bacteria that can solve some of these problems.”
The research was supported by: state funding to the UW-Madison College of Agricultural and Life Sciences, and the College of Engineering; a USDA Hatch grant from the College of Agricultural and Life Sciences; and a National Science Foundation Early Career Development Award to Brian Fox.