A can of spoiled meat and nuclear waste may appear to have little in common, but the microbe Deinococcus radiodurans finds both environments rather cozy. Scientists hope this organism's ability to withstand massive doses of radiation will make it a useful tool for toxic-site remediation.
Although scientists now find it in many different soil and water sites around the world, D. radiodurans was not identified until 1956. It was isolated from a can of ground beef that had been radiation sterilized but had spoiled nonetheless. Perhaps because it can efficiently repair radiation breakage of its own DNA, D. radiodurans can endure 1.5 million rads of radiation, a dose 3000 times higher than would kill organisms from microbes to humans. Scientists are unsure how this resistance evolved, although they suspect it may be a side effect of the microbe's ability to survive periods of severe dehydration, which also fragments DNA.
Recognition of D. radiodurans' resistance to radiation led DOE Microbial Genome Program (MGP) managers to believe the microbe could be useful in cleaning up mixed-waste sites contaminated with toxic chemicals as well as radiation. They began to fund projects to decipher the microbe's genome and alter it to detoxify the most common chemical contaminants at these sites. Such detoxification functions might include concentrating heavy metals and breaking down organic solvents such as trichlorethylene.
Some results are reported below.
Complete Genome Sequence The complete sequence of the 3-Mb D. radiodurans genome is now in hand, and researchers led by Owen White at The Institute for Genomic Research (TIGR) in Rockville, Maryland, expect to publish their findings shortly. The genome consists of three chromosomes and a single extrachromosomal plasmid, with repeats highly abundant on each chromosome. Circularization of chromosomal regions, occurring across repeats distributed at least every 50 kb, may be part of the homologous recombination system that is the major form of repair for DNA double-strand breaks. Researchers have not yet determined if circularization occurs more frequently after irradiation. No evidence, however, exists for a causal link between circularization and radiation resistance; the bacterium Escherichia coli's genome, in fact, also circularizes and yet is radiation sensitive. Plausible explanations for the extraordinary DNA-repair capability of D. radiodurans remain elusive in the early analyses of DNA repair genes.
In the sequencing effort, assembly problems were encountered in repeated regions over 500 bases long and more than 95% identical. To help verify the assemblies, TIGR scientists turned to a special type of "optical" chromosome map of D. radiodurans constructed by David Schwartz and colleagues [New York University (NYU)].
To create this type of map, the NYU team uses optical light microscopy to directly image individual DNA molecules bound to specially coated surfaces, which are then cut with restriction enzymes. When a cut is made, the linear DNA contracts and reveals a break. Scientists create a landmark map of the DNA sequence by determining where the cut sites lie and then measuring the distances between them. This type of high-resolution restriction enzyme map provides a useful scaffold for aligning and verifying the maps predicted by standard shotgun-sequencing procedures.
Optical mapping of D. radiodurans, which is providing insight into this organism's biology with a picture of the entire genome's basic organization, also may help scientists understand aspects of the microbe's radiation-resistant nature.
Genetic Enhancements Cleanup of toxic sites created by improper disposal of nuclear wastes presents a massive global challenge requiring innovative remediation approaches. In Nature Biotechnology (Vol. 16, October 1998), DOE grantees Michael Daly and Kenneth Minton (Uniformed Services University for the Health Sciences in Bethesda, Maryland) described a first step toward enhancing the D. radiodurans genome to make it valuable for toxic-site cleanup. The work also was featured in a four-page "Conan the Bacterium" article in the July-August, 1998, issue of The Sciences, the magazine of the New York Academy of Sciences.
In the Nature Biotechnology article, Daly and Minton reported sucessfully altering the microbe's genome. This was accomplished by first fusing a gene encoding toluene dioxygenase (an enzyme that degrades the organic contaminant toluene) to a D. radiodurans promoter (a site that activates the gene). This DNA was then inserted into one of the bacterium's chromosomes. The resulting recombinant bacterium is capable of degrading toluene and other organic compounds in a high-radiation environment. It also is tolerant of toluene and trichloroethylene's solvent effects at levels exceeding those of many radioactive waste sites.
