University Of Cincinnati Biologists Find Evidence For Unusual DNA Repair InArchaea

May 20, 1998

CINCINNATI, Ohio -- Microbes that survive in extreme environments might have a much higher rate of DNA recombination than other prokaryotes according to research which will be presented Wednesday, May 20 during the annual meeting of the American Society for Microbiology.

Scientists know that Sulfolobus acidocaldarius grows optimally around 80 degrees Celsius and in hot, sulfur springs with a pH of about 3. They know very little else about the microbe or any other member of the archaea, the so-called third domain of life.

"We have amassed a lot of knowledge about the cellular functions of eukaryotic cells and bacteria...then we get to the archaea which do not share many properties with the other groups, and we know almost nothing about them," said Dennis Grogan, assistant professor of biological sciences at the University of Cincinnati. "The ratio of knowledge is something like 10,000 to one."

Grogan is working to understand the genetics of Sulfolobus acidocaldarius. In previous work, Grogan demonstrated that the microbe can use visible light to repair DNA damaged by ultraviolet light. Now, he is searching for tools to study genetic recombination in Sulfolobus.

"Because they are so different, you can't necessarily take experimental methods, especially genetic methods, and transfer things that were done in E. coli (a common intestinal bacterium) and apply them to these organisms."

Grogan began his most recent work by using Sulfolobus mutants in genetic recombination experiments. A common type of microbial mutant is called an auxotroph or nutritional mutant. The mutant requires a supplement in its growth medium to replace a particular nutrient a normal cell could produce on its own. For example, one mutant might need the amino acid histidine while another requires tryptophan in its diet. Finding these mutants was a chore, to say the least.

"The amino acid mutations are hard to get. We checked roughly 15-thousand mutagenized colonies to find about three dozen useful auxotrophic mutants. These things are hard to get."

In contrast, Grogan and his graduate students found it was very easy to recover mutations in another gene known as pyrF. PyrF mutants require uracil, but Grogan has been able to use a genetic technique called selection to isolate them easily. So far, his student Michelle Reilly has found more than 200 different pyrF mutants.

Once Grogan had cells with more than one kind of mutation, he and his students set up classical genetic recombination experiments. It appears Sulfolobus is able to exchange genetic material with other cells through a process known as conjugation. This is an effective way to map genes on an E. coli chromosome, but so far, it hasn't worked out very well with Sulfolobus. "The long-range mapping doesn't appear to be panning out, and that might tell us something about the frequency of recombination. If the frequency of recombination is very high, then that's one way we can explain our results."

In contrast, looking for recombination with the single pyrF gene produced very different and much more clear-cut results. Reilly looked for recombination between cells with different mutations within the same pyrF gene. "You find that it's very probable if you take two of these mutants, you can isolate recombinants," said Grogan. "About 95 percent of the mutations are far enough part in this one gene that they will give you a number of recombinants."

That indicates a very high rate of recombination in Sulfolobus. So, Grogan can't be sure if the pyrF recombinants are being formed by classical genetics or not. "It might not be recombination in the classical sense that you make a reciprocal crossover. There are other types of recombination that are considered part of DNA repair."

The DNA repair hypothesis makes a lot of sense to Grogan, because the background mutation rate in Sulfolobus is nearly identical to E. coli, even though Sulfolobus lives in an environment much more likely to damage DNA. "They're oxidizing sulfur to produce sulfuric acid. That's how they generate energy. So, they're in this acidic environment and it's always very hot. Those conditions are death to most proteins and nucleic acids from other organisms."

In fact, Grogan's rough estimate indicates Sulfolobus DNA is at 1,000 times higher risk of damage than E. coli. His work has been supported by the U.S. Office of Naval Research.

University of Cincinnati

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