HHMI researcher Ronald Breaker of Yale University has discovered the cellular chain of events that occurs inside a bacterium after it encounters fluoride in its environment. His team's findings reveal that many bacteria try to fend off fluoride – which the organisms treat as a toxic substance – by throwing it out. The presence of such a transport system indicates that fluoride itself has antimicrobial properties, Breaker said. The findings are published online in Science Express on December 22, 2011.
Breaker's lab studies non-coding RNA, stretches of genetic material that play regulatory roles in the cell instead of coding for proteins. Using different computer algorithms, he and his colleagues analyze the genomes of organisms to identify signature sequences in genetic material that likely indicate the presence of noncoding RNA. Among the types of non-coding RNAs they find are regulatory molecules called riboswitches. Normally, the role of a riboswitch is easy to deduce: Riboswitches are attached to the genes that they regulate. If the gene is needed to produce a certain compound, the riboswitch is usually sensitive to that compound, so when its level increases or decreases in the cell, the riboswitch can cause more or less to be made. Aside from their interest in the biology of riboswitches, Breaker's group is studying these genetic switches because they could represent new drug targets and might be used to control the activity of genes inserted into cells as gene therapies.
In a recent set of experiments, Breaker's team identified a new riboswitch that was attached to a handful of genes with vague or unknown functions. They were stumped. "We knew we had a riboswitch but we didn't know what it bound," says Breaker. "And we were very intrigued because it was one of the only non-coding RNAs we've ever found that's present in both bacteria and archaea. That suggests that it has ancient origins and an important role in the cell," he notes.
So Breaker and his colleagues put the RNA in a test tube and began to mix in different chemicals, observing whether or not they bound to the riboswitch. They worked through a long list of more common chemicals before they stumbled on fluoride. The addition of fluoride was an accident -- fluoride was a contaminant in a sample of a different chemical they were testing.
Once Breaker's group found that the riboswitch bound to fluoride, they were able to show that the genes controlled by the riboswitch are those that help the cell fight the toxicity of fluoride. Fluoride, a negatively charged ion, binds aggressively to some metabolites and essential enzymes. If fluoride floods a cell, it can quickly shut down cellular processes. The fluoride-sensing riboswitch, Breaker's team discovered, turns on a gene coding for ion channels that transport fluoride back out of the cell.
"This riboswitch is detecting fluoride buildup in the cell and turning on genes to help overcome that buildup," says Breaker. Whether or not the riboswitch is successful, and fast enough, determines whether a bacterium can fight the effects of fluoride.
"Our data not only help explain how cells fight the toxicity of fluoride, but it also gives us a sense of how we might be able to enhance the antimicrobial properties of fluoride," says Breaker. "In the future we might be able to use this knowledge to make fluoride even more toxic to bacteria." Blocking the fluoride channel, for example, makes cells 200 times more sensitive to fluoride, the researchers showed. Finding other ways to enhance fluoride's effects—by inactivating the riboswitch or shutting off other downstream genes—could make fluoride an even better antimicrobial agent.
Jim Keeley | EurekAlert!
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