Thermus aquaticus

Thermophilus aquaticus is a species of bacterium that can tolerate high temperatures; it is the source of the heat-resistant enzyme Taq DNA Polymerase, one of the most important enzymes in molecular biology because of its use in the polymerase chain reaction. Thermophilus aquaticus is one of several thermophilic bacteria that belong to the Deinococcus-Thermus group.

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History

When studies of biological forms in the Yellowstone hot springs began in the 1960s, scientists thought that the life of thermophilic bacteria could not be sustained in temperatures above about 55 degrees Celsius (131 degrees Fahrenheit). Soon, however, it was discovered that many bacteria in different springs not only survived but also thrived in higher temperatures. In 1969, Thomas Brock and Hudson Freeze of Indiana University reported a new species of thermophilic bacterium which they named Thermophilus aquaticus.^[1] The bacterium was first discovered in the Great Fountain region of Yellowstone National Park and has since been found in similar thermal habitats around the world.

Biology

It thrives at 70 degrees Celsius (160 degrees Fahrenheit), but can survive at temperatures of 50 to 80 °C (120 to 175 °F).This bacteria is a chemotroph, meaning that it performs chemosynthesis in order to obtain food. However, since its range of temperature overlaps somewhat with that of the photosynthetic cyanobacteria that share its ideal environment it is sometimes found living in conjuncture with its neighbors, obtaining energy for growth from their photosynthesis.

Enzymes from T. aquaticus

T. aquaticus was to eventually become famous as a source of thermostable enzymes, particularly the "Taq" DNA Polymerase, as described below.

Aldolase

Studies of this extreme thermophilic bacterium that could be grown in cell culture was initially centered on attempts to understand how protein enzymes (which normally inactivate at high temperature) can function at high temperature in thermophiles. In 1970 Freeze and Brock published an article describing a thermostable aldolase enzyme from Thermophilus aquaticus.^[2]

RNA polymerase

The first polymerase enzyme isolated from Thermophilus aquaticus was a DNA-dependent RNA polymerase (G.M. Air and J.I. Harris, 1974). This was the year that DNA sequencing was made practical by the invention of the labeled-chain termination method.^[3] DNA sequencing typically depends on the use of DNA-directed DNA polymerases, enzymes that make a new strand of DNA starting with an existing strand. "Primed synthesis" refers to the fact that DNA polymerases usually need a region of DNA double helix as a start point for attaching to the DNA strand that is to be replicated. In the test tube, the start point can be determined by providing a primer, a short strand of DNA that will attach by base pairing to the target strand.

DNA polymerase ("Taq pol")

For more details on this topic, see Taq polymerase.

DNA polymerase was first isolated from Thermophilus aquaticus in 1976.^[4] The first advantage that was found for this thermostable (temperature optimum 80°C) DNA polymerase was that it could be isolated in a purer form (free of other enzyme contaminants) than could the DNA polymerase from other sources. A molecular model of the structure of this enzyme is shown to the right.

Taq I restriction enzyme

For more details on this topic, see TaqI.

Most molecular biologists probably became aware of Thermophilus aquaticus in the late 1970s or early 1980s because of the isolation of useful restriction endonucleases from this organism.^[5]

Note: The term "Taq" to refer to Thermophilus aquaticus arose from the convention of giving restriction enzymes short names such as Sal and Hin, names that come from the genus and species names of the source organisms.

Works of Kary Mullis

In the early 1980s Kary Mullis was working at Cetus on the synthesis of DNA. There was interest there in developing methods for detecting gene mutations that would be useful in disease screening. A major problem was that the available techniques (such as oligomer restriction) relied upon having a lot of DNA copies of the mutated gene. Mullis was familiar with the idea of using DNA oligonucleotides and hybridizing them to target DNA strands.

Idea Number One: PCR

In 1983, to advance a project he was involved with, Mullis began to consider using two oligonucleotides, one to hybridize to each strand of a DNA double helix. At first, his only reason for adding DNA polymerase to his experiments was as a way of making sure that deoxynucleoside triphosphates would be removed from his samples. But he then realized that the enzyme might make useful copies of the oligonucleotide-primed DNA strands. He immediately realized this was a potential way to amplify a region of DNA.^[6]

Note: If you are not familiar with the polymerase chain reaction procedure, you may want to read about it before continuing with this article.

The main problem was, that after a round of strand copying, the DNA would have to be heated to near boiling in order to denature the newly formed double stranded DNA, allowing the strands to separate and open new templates for another round of amplification. This heating step would denature and inactivate the DNA polymerase, requiring that new enzyme be added at each amplification step.

This original PCR technique was slow and labor-intensive. The "inside-the-box" thinkers at Cetus began to automate the process. The first PCR machine, "Mr. Cycle" automatically added more enzyme after every heating and cooling step.

Idea Number Two: Thermostable Taq Polymerase

Those who know Mullis, such as Thomas J. White, agree that it was Mullis who came up with the idea of using Taq polymerase in order to avoid having to add polymerase to the PCR reaction during the thermocycling process. This was the key idea that made the PCR technique available to an army of molecular biologists.

Roche Molecular Systems eventually bought the PCR patents from Cetus for $300,000,000. Kary Mullis got $10,000 from Cetus and a Nobel Prize from his scientific peers. Research scientists and biotechnology companies spend hundreds of millions of dollars each year for Taq polymerase.

A 1988 article describing the use of Taq polymerase for PCR^[7] led to Science magazine naming Taq polymerase its first "Molecule of the Year" in 1989.

By 1989 the PCR technique was being used in all areas of modern biology research, including in clinical research. It began to find a pressing application in AIDS detection.^[8]

See also

• Bacteria
• Thermophiles
• Geyser

Figure: PCR Publications by Year.

The PCR technique became a major tool for molecular biology after the Taq DNA Polymerase and thermocyclers became available.

References

1. ^ http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=5781580
2. ^ Freeze; Brock (1970). "{{{title}}}".
3. ^ Sanger, Frederick; Donelson JE, Coulson AR, Kossel H, Fischer D. "Determination of a nucleotide sequence in bacteriophage f1 DNA by primed synthesis with DNA polymerase". Journal of Molecular Biology 5 90 (2): 315–33.
4. ^ Chien, A; D B Edgar, and J M Trela. "Deoxyribonucleic acid polymerase from the extreme thermophile Thermophilus aquaticus". Journal of Bacteriology 127 (3): 1550–1557.
5. ^ Sato, S. (Feb 1978). "A single cleavage of Simian virus 40 (SV40) DNA by a site specific endonuclease from Thermophilus aquaticus, Taq I". J. Biochem (Tokyo) 83 (2): 633–5.
6. ^ Saiki, RK; Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N (Dec 20 1985). "Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia". Science 230 (4732): 1350-4.
7. ^ Saiki, RK; Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA (1988). "Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase". Science 239: 487–91.
8. ^ Guatelli, J C; T R Gingeras, and D D Richman (1989). "Nucleic acid amplification in vitro: detection of sequences with low copy numbers and application to diagnosis of human immunodeficiency virus type 1 infection". Clin Microbiol Rev. 2: 217–226.