Thermus aquaticus
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| Thermus aquaticus | ||||||
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Description and significance
. Thermus Aquaticus isolated in 1969 by brocks and Freeze of University of Indiana. It is a gram negative bacteria both motile (presence of a flagellum) or immotile. Comparisons between structures of Thermus Aquaticus and E. Coli have shown many similarities linking them to a common ancestor. YT-1 gene extracted from Thermus Aquaticus is structurally similar to Rec A from E. Coli homologue and comaparisons between Klentaq1, a large fragment of Taq DNA polymerase and the Klenow fragment of E Coli DNA polymerase reveal identical C terminals and very similar N terminals. Discovery of this species of thermophiles primarily came as a surprise to researchers and very soon proved to be and extremely important tool in many fields of sciences including biology, microbiology, genetics, diagnostics, clinical laboratories, forensic and environmental sciences, hereditary studies and paternity testings. The importance of this discovery comes from the high thermostability of the Thermus Aquaticus proteins. The taq polymerase plays an extremely important role in Polymerase Chain Reaction. PCR is an amplification process of one or a few copies of DNA using thermal cycling introduced by Kary Mullis in 1984 winning him the Noble peace prize in 1993. The enzymes found in the Thermus Aquaticus are able to withstand the heat in the denaturing of the newly formed DNA so strands can seperate and act as templates for the next cycle of PCR. The Taq polymerase with an optimum activity at 72-80 degrees celsius and a half life of 9 minutes at 97.5 degrees celsius can replicate 9000 base pairs in less than 10 seconds, replaced DNA polymerase from E. Coli and presently still plays an extremely important role in gaining insight into realm of biotechnology.
Genome structure
Thermus Aquaticus contains pillus like structures used in conjugation. 12 genes in 3 loci were found to encode for preplin like proteins essential for natural transfomation. It has double stranded circular DNA with a length of 2,338,193 nt. with a replicon type WGS, (Master Wgs), no pseudogenes, 53 structural RNA's, 1982 protein codings, and pTT27 plasmid.
Cell structure and metabolism
Thermus Aquaticus not only can function at high temperatures but they thrive in higher temperatures. Optimum growth is seen between 60 and 75 degrees celsius (could go as low as 35 degrees celsius to as high as 85 degrees celsius). PH ranges from 7.5 to 8.0 generally but some strains could be between 5.1 to 9.5. This organism is a chemotroph using carbohydrates, amino acids, caboxylic acids and peptides for growth. Monosaccharides are generally used for carbons sources but sucrose, maltose(Icelandic stains) and even some fewer strains use glucose. There is also a variance in the proteins isolated from different strains; Elastin, Fibrin and Casein. Not all strains can hydrolize all substrates. Membranes of these proteins are remarkably temperature stable and the heat stability of the enzymes and proteins synthesis systems allow them to function efficiently at high tempratures. Many factors contribute to the stability of the proteins:
1) Highly organized hydrophobic interiors 2) More hydrogen bonds and presence of other non covalent bonds strengthen the structure. 3) Larger quantities of amino acids like Proline make peptide chain less flexible. 4) Proteins aided and stabilized by special chaperone proteins. 5) Some evidence that DNA is stabilized by histone like proteins. 6) Their membrane lipids tend to be more saturated and more branched and posses higher molecular weight resulting in a higher melting point and in turn more thermostable.
Ecology
Thermus Aquaticus was first isolated in the Great Fountain region of Yellowstone National Park from neutral and alkaline springs in 1969 by Brocks and Freeze. This discovery disproved the previous beliefs that bacteria could not function properly at higher temperatures. After this discovery, some strains of Thermus Aquaticus were discovered in hot springs in Iceland and hydrothermal vents in other parts of the world.
CONTROVERSY: After the isolation of Thermus Aquaticus, samples of it were deposited in the American Type Culture Collection, which is a public repository and other scientists were able to obtain samples and do more research. By about 1980's it was pretty obvious the potential for commercialising the enzymes from this organism were extremely high and lots of profits and revenue were involved. A Swiss pharmacuedical copany called Laroche patened it and so the National Park Systems were not receiving any; of the profits eventhough the organism was isolated at a national park which is public property and so this is called "great Taq ripoff". Since then researches at the parks sign an agreement of "benefits sharing" so portions of profits can get back to park services.
The fight for patent right's of the Taq enzymes are still going on. A Swiss company "Roche Corporation" is claiming patent rights but European Patent office is rejecting their claims.
Pathology
Thermus Aquaticus has not been associated with any known pathology.
Application to Biotechnology
Enzymes derived from Thermus Aquaticus have had an incredibly important role in facilitating many aspects of biotechnology correlated with DNA amplification and enabling researchers to study proteins and enzymes under conditions not possible before and this is all due to the thermostability of the protiens and there ability to function at even higher rates at high temperatures. Some of the isolated enzymes and their roles in facilitaitng applications in biotechnology are as follows:
1) Adolase- a themostable enzyme (protein enzyme that functions well at high temperature. 2) RNA polymerase- first polymerase isolated from Taq in1974. 3) Restriction Endonucleases 4) DNA polymerase- isolated in 1976, could be isolated in purer form and later discoveered to be used in PCR, for amplifying short segments of DNA (before the discovery of the Taq DNA, enzymes needed to be added after each cycle of denaturing of DNA, but with the use of the Taq DNA polymerase it was not necessary anymore.) One single copy of genomic sequence was amplified by a factor of more than 10 million(One ng of DNA template was amplified up to 35 kb and target DNA molecule present only once in a sample of 105 cells) with improved base pair fidelity and the PCR product used as primers for maximum yield. The specificity , sensitivity , yield and length of product could be amplified. This enzyme was soon cloned, sequenced, and produced in mass quantities for commercial sale. 5) Other enzymes with high optimal temperatures allowing researchers to study them in extreme conditions are:DNA ligase, Alkaline Phosphotase, NADH Oxidase, Isocitrade, Dehydrogenase, Amylomaltase and Fructose1,6-Biophosphate-Dependedent L Lactate Dehydrgenase.
Besides the revolutionary changes in PCR, LCR, using Taq Ligase can amplify genetic sequences of stretches of DNA that posses a desired sequence million or more times within hours. It can amplify and screen in a single step and screen for mutations simultanously. LCR is useful in testing for hereditary diseases, revealing hidden infections and distinguishing between drug resistant and drug sensitive strains of viruses and bacteria .
Current Research
Recent studies have emphasized the role of disulfide bonds in stabilizing structure of intracellular proteins of Thermus Aquaticus among some other thermophiles. Previously the popular belief was disulfide bonds are only present in extracellular proteins where they stabilize folded proteins against harsh conditions and are rarely found in the cytosil. The specific protein which seems to be responsible for the formation of intracellular disulfide bonds seems to be Protein Disulfide Oxiidoreductase(PDO), which functions as a cytoplasmic PDI. It has been suggested that Eucaryotic enzyme PDI, found in the Endoplasmic Riticulum where it catalyzes isomerization of protein disulfide bonds, has evolved from a protein similar to thermophilic PDO. More research needs to be done on this subject.
Besides thermophiles, elevated intracellular disulfide bonding has been seen in other extremophiles including; halophiles, alkalophiles, acidophiles, and radiant tolerant organisms. This supports the role of intracellular disulfide bonds in stabilizing proteins in all types of extreme conditions.
This study sheds some light on different methods used by organisms to stabilize their proteins to adapt to "exotic" environments.
References
Barnes,W.M. PCR amplification of up to 35 kb DNA with high fidelity and high yield from bacteriophage templates-proceedings of the National Academy of Sciences of the United States of America v.91(March 15,1994)p.2216-20
Weiss, R. Hot prospect for new gene amplifier-Science v.254(November 29,1991)p.1292-3
Saiki, R.K.,et.al., Primer directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science v.239 (January 29,1988)p.487-91.
<http://microbewiki.kenyon.edu/index.php/Thermus> The Genomics of Disulfide Bonding and Protein Stabilization in Thermophiles Beeby M, O'Connor BD, Ryttersgaard C, Boutz DR, Perry LJ, et al. PLoS Biology Vol. 3, No. 9, e309 doi:10.1371/journal.pbio.0030309