Scientists at the U.S. Department of Energy’s Brookhaven National Laboratory have developed a new, high-throughput technique for identifying the many species of microorganisms living in an unknown “microbial community.” The method, described in the March 2006 issue of Applied Environmental Microbiology, has many applications — from assessing the microbes present in environmental samples and identifying species useful for cleaning up contamination to identifying pathogens and distinguishing harmless bacteria from potential bioterror weapons.
“Microbial communities are enormously diverse and complex, with hundreds of species per milliliter of water or thousands per gram of soil,” said Brookhaven biologist Daniel (Niels) van der Lelie, lead author of the study. “Elucidating this complexity is essential if we want to fully understand the roles microbes play in global cycles, make use of their enormous metabolic capabilities, or easily identify potential threats to human health.”
Growing cultures of microbes to identify species is slow and error prone as the culture conditions often screen out important members of the community. Sequencing entire genomes, while highly specific and informative, would be too labor intensive and costly. So scientists have been searching for ways to identify key segments of genetic code that are short enough to be sequenced rapidly and can readily distinguish among species.
The Brookhaven team has developed just such a technique, which they call “single point genome signature tagging.” Using enzymes that recognize specific sequences in the genetic code, they chop the microbial genomes into small segments that contain identifier genes common to all microbial species, plus enough unique genetic information to tell the microbes apart.
In one example, the scientists cut and splice pieces of DNA to produce “tags” that contain 16 “letters” of genetic code somewhat “upstream” from the beginning of the gene that codes for a piece of the ribosome — the highly conserved “single point” reference gene. By sequencing these tags and comparing the sequenced code with databases of known bacterial genomes, the Brookhaven team determined that this specific 16-letter region contains enough unique genetic information to successfully identify all community members down to the genus level, and most to the species level as well.
“Sequencing is expensive, so the shorter the section you can sequence and still get useful information, the better,” van der Lelie said. “In fact, because these tags are so short, we ‘glue’ 10 to 30 of them together to sequence all at one time, making this a highly efficient, cost-effective technique.”
For tag sequences that can’t be matched to an already sequenced bacterial genome (of which there are only a couple hundred), the scientists can use the tag as a primer to sequence the entire attached ribosomal gene. This gene is about 1400 genetic-code-letters long, so this is a more time-consuming and expensive task. But since ribosomal genes have been sequenced and cataloged from more than 100,000 bacterial species, this “ribotyping” technique makes use of a vast database for comparison.
“If there’s still no match,” said van der Lelie, “then the tag probably identifies a brand new species, which is also very interesting!”
In another test with possible applications for identifying agents used in bioterror attacks, the technique also clearly discriminated between closely related strains of Bacillus cereus, a pathogenic soil microbe, and Bacillus anthracis, the bacterial cause of anthrax.
This technique could also help assess how microbial community composition responds to changes in the environment. Such information might help identify which combinations of species would be best suited to, say, sequestering carbon or cleaning up radiological contamination.
This study represents just one application of genome signature tagging, a technique developed at and patented by Brookhaven Lab. Brookhaven scientists have also used genome “tags” to identify the sites where regulatory proteins bind to DNA (more) . This research could greatly speed the process of unraveling the role these proteins play in turning on and off certain genes in different types of cells — as well as what might go awry in conditions like cancer.
This research was funded by the Office of Biological and Environmental Research within the U.S. Department of Energy’s Office of Science and by Brookhaven’s Laboratory Directed Research and Development funds.
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