| Pyrococcus furiosus | |
|---|---|
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| Pyrococcus furiosus | |
Scientific classification | |
| Domain: | Archaea |
| Kingdom: | Methanobacteriati |
| Phylum: | Methanobacteriota |
| Class: | Thermococci |
| Order: | Thermococcales |
| Family: | Thermococcaceae |
| Genus: | Pyrococcus |
| Species: | P. furiosus |
| Binomial name | |
| Pyrococcus furiosus Fiala and Stetter, 1986 | |
Pyrococcus furiosus is aheterotrophic, strictlyanaerobic,extremophilic,model species ofarchaea. It is classified as ahyperthermophile because it thrives best under extremely high temperatures, and is notable for having an optimum growth temperature of 100 °C (a temperature that would destroy most living organisms).[1]P. furiosus belongs to thePyrococcus genus, most commonly found in extreme environmental conditions ofhydrothermal vents. It is one of the fewprokaryotic organisms that hasenzymes containingtungsten, an element rarely found in biological molecules.
Pyrococcus furiosus has many potential industrial applications, owing to its uniquethermostable properties.P. furiosus is used in the process of DNA amplification by way ofpolymerase chain reaction (PCR) because of its proofreading activity. UtilizingP. furiosus in PCR DNA amplification instead of the traditionally usedTaq DNA polymerase allows for a significantly more accurate process.[2] The thermodynamic stability ofP. furiosus' enzymes is useful in the creation of diols for laboratory and industrial purposes. Certainsuperoxide dismutases found inP. furiosus can be introduced into plants to increase their tolerance in environmentally stressful conditions and ultimately their survival.[3]
Pyrococcus furiosus is astrictly anaerobic,heterotrophic, sulfur-reducing archaea originally isolated from heated sediments inVulcano, Italy by Fiala and Stetter. It is noted for its rapid doubling time of 37 minutes under optimal conditions, meaning that every 37 minutes the number of individual organisms is multiplied by two, yielding an exponential growth curve. Each organism is surrounded by a cellular envelope composed ofglycoprotein called anS-layer. It appears as mostly regularcocci—meaning that it is roughly spherical—of 0.8 μm to 1.5 μm diameter with monopolar polytrichousflagellation.[1]
Aglycoprotein notable to archaea species makes up the majority of the composition ofP. furiosusflagella. Aside from potentially using them for swimming, these flagella were observed under lab conditions in use for unique applications such as forming cell to cell connections during stationary growth phase. They are additionally utilized as cable-like connectors to adhere to various solid surfaces such as sand grains in the habitat in which this species was discovered. This may lead to the formation of microcolonialbiofilm-like structures.
P.furiosus grows between 70°C (158°F) and 103 °C (217 °F), with an optimum temperature of 100 °C (212 °F), and between pH 5 and 9 (with an optimum at pH 7). Since it uses fermentation of carbohydrates, it grows well on yeast extract,maltose,cellobiose, β-glucans,starch, and protein sources (tryptone, peptone, casein, and meat extracts) through the Embden-Meyerhoff pathway. This is a relatively wide range of sources when compared to other archaea. Growth is very slow, or nonexistent, on amino acids, organic acids, alcohols, and most carbohydrates (includingglucose,fructose,lactose, andgalactose). The metabolic products ofP. furiosus areCO2 andH2. The presence of hydrogen severely inhibits its growth and metabolism; this effect can be circumvented, however, by introducingsulfur into the organism's environment. In this case,H2S can be produced through its metabolic processes seemingly for the purpose of detoxication or energy conservation, not energy production. While many other hyperthermophiles depend on sulfur for growth,P. furiosus does not.[4]
P. furiosus is also notable for an unusual and intriguingly simple respiratory system, which obtains energy by reducing protons to hydrogen gas and uses this energy to create an electrochemical gradient across its cell membrane, thereby drivingATP synthesis. This could be a very early evolutionary precursor of respiratory systems in all higher organisms today.[5]
The sequencing of the completegenome ofPyrococcus furiosus was completed in 2001 by scientists at theUniversity of Maryland Biotechnology Institute. The Maryland team found that the genome has 1,908 kilobases, including 2,065open reading frames (ORFs) that encode proteins.[6] A study performed in 2005 revealed 17 new ORFs specific toPyrococcus furiosus that were not originally annotated, bringing the number of ORFs up to 2,082.[7]
A lab strain ofPyrococcus furiosus named COM1 is commonly used for its "high plasticity" and ability to take up foreign DNA, owing to its highrecombination andtransposon activity. It has 1,571 more base pairs than the referenced NCBI genome, and 10 moreinsertion sequences (ISs). These ISs have deactivated 13 genes and many more are altered, but the strain's growth is yet comparable to its parent strain.[8]
Pyrococcus furiosus possesses several highly thermostablealcohol dehydrogenases (ADHs): the short-chain AdhA, the iron-containing AdhB, the zinc-containing AdhC, and more.[9][10] Each of these ADHs are NADP-dependent, and serve to replenish NADP+ by using the NADPH produced byfermentation to reduce aldehydes to alcohols. The aldehydes are also products of fermentation and are toxic to the cell, so removal is necessary.P. furiosus ADHs typically have a broad range of aldehyde substrates they can use, and they can also catalyze the reverse reaction (oxidation of alcohols) using ethanol, 1,3-propanediol, and other alcohols for substrate. As with most of the archaea's enzymes, the ADHs are sensitive to oxygen.[11]
Pyrococcus furiosus has five unique tungsten-containingoxidoreductases that are part of its NAD(P)H-independentglycolytic pathway. These enzymes function optimally above 90 °C. The first to be discovered wasaldehydeferredoxin oxidoreductase, or AOR, which utilizes tungsten, sulfur, and iron to catalyze theoxidation of aldehydes and reduce ferredoxin (this being theelectron carrier instead of NAD(P)H).[12] As this was the first, all tungsten-containing oxidoreductases are said to be part of the AOR family. The next oxidoreductase to be discovered wasglyceraldehyde-3-phosphate ferredoxin oxidoreductase, or GAPOR, which utilizes tungsten and iron to catalyze the oxidation of specifically glyceraldehyde-3-phosphate. GAPOR only functions under anaerobic conditions, as with many enzymes inP. furiosus.[13] Another oxidoreductase isformaldehyde ferredoxin oxidoreductase, or FOR, which catalyzes the oxidation of aldehydes intocarboxylic acids. This enzyme utilizes four types of cofactors: tungsten, iron, sulfur, and calcium.[14] The next oxidoreductase, WOR4, does not help oxidize aldehydes, but rather has a role in the reduction of elemental sulfur (S0) into H2S. This uses the same cofactors as FOR, and is only found in P. furiosus cells that are grown in the presence of elemental sulfur.[15] The fifth and final oxidoreductase is named WOR5, and it has a broad specificity foraromatic andaliphatic aldehyde species.[16]
An oxidoreductase species inP. furiosus that does not contain tungsten ispyruvate ferredoxin oxidoreductase, or POR, which catalyzes the final step of the glycolytic pathway. It is possible that POR is an ancestor of mesophilic pyruvate oxidoreductases.[17] There is also the indolepyruvate ferredoxin oxidoreductase, or IOR, which utilizes iron and sulfur to catalyze the "oxidativedecarboxylation ofaryl pyruvates."[18]
ADNA polymerase was discovered inP. furiosus that was thought to be unrelated to other known DNA polymerases, as no significantsequence homology was found between its two proteins and those of other known DNA polymerases. This DNA polymerase has strong3'-to-5' exonucleolytic activity and a template-primer preference which is characteristic of a replicative DNA polymerase, leading scientists to believe that this enzyme may be the replicative DNA polymerase ofP. furiosus.[19] It has since been placed in the family B of polymerases, the same family as DNA polymerase II. Its structure, which appears quite typical for polymerase B, has been solved as well.[20][21]
Since theenzymes ofP. furiosus are extremely thermostable, theDNA polymerase fromP. furiosus (also known asPfu DNA polymerase) can be used in thepolymerase chain reaction (PCR) DNA amplification process. The PCR process must use a thermostable DNA polymerase for automated in vitro amplification, which was originally used Taq DNA polymerase.[22] However, since purifiedTaq DNA polymerase lacksexonuclease (proofreading) activity, it cannot excise mismatchednucleotides. Researchers discovered in the early 1990s that thePfu DNA polymerase ofP. furiosus does possess a requisite3’-to-5’ exonuclease activity allowing for the removal of errors. Subsequent tests utilizingPfu DNA polymerase in the PCR process revealed a more than tenfold improvement over the accuracy of usingTaq DNA polymerase.[2]
One practical application ofP. furiosus is in the production ofdiols for various industrial processes. It may be possible to use the enzymes ofP. furiosus for applications in such industries as food, pharmaceuticals, and fine-chemicals in whichalcohol dehydrogenases are necessary in the production of enantio- and diastereomerically pure diols. Enzymes from hyperthermophiles such asP. furiosus can perform well in laboratory processes because they are relatively resistant: they generally function well at high temperatures and high pressures, as well as in high concentrations of chemicals.
In order to make naturally derived enzymes useful in the laboratory, it is often necessary to alter their genetic makeup. Otherwise, the naturally occurring enzymes may not be efficient in an artificially induced procedure. Although the enzymes ofP. furiosus function optimally at a high temperature, scientists may not necessarily want to carry out a procedure at 100 °C (212 °F). Consequently, in this case, the specific enzyme AdhA was taken fromP. furiosus and put through various mutations in a laboratory in order to obtain a suitable alcohol dehydrogenase for use in artificial processes. This allowed scientists to obtain a mutant enzyme that could function efficiently at lower temperatures and maintain productivity.[23]
The expression of a certain gene found inP. furiosus in plants can also render them more durable by increasing their tolerance for heat. In response to environmental stresses such as heat exposure, plants producereactive oxygen species which can result in cell death. If these free radicals are removed, cell death can be delayed. Enzymes in plants calledsuperoxide dismutases removesuperoxide anion radicals from cells, but increasing the amount and activity of these enzymes is difficult and not the most efficient way to go about improving the durability of plants.[24]
By introducing thesuperoxide reductases ofP. furiosus into plants, the levels of O2 can be rapidly reduced.[citation needed] Scientists tested this method using theArabidopsis thaliana plant. As a result of this procedure, cell death in plants occurs less often, therefore resulting in a reduction in the severity of responses to environmental stress. This enhances the survival of plants, making them more resistant to light, chemical, and heat stress.
This study could potentially be used as a starting point to creating plants that could survive in more extreme climates on other planets such as Mars. By introducing more enzymes from extremophiles likeP. furiosus into other species of plants, it may be possible to create incredibly resistant species.[3]
By comparingP. furiosus with a related species of archaea,Pyrococcus abyssi, scientists have tried to determine the correlation between certain amino acids and affinity for certain pressures in different species.P. furiosus is notbarophilic, whileP. abyssi is, meaning that it functions optimally at very high pressures. Using two hyperthermophilic species of archaea lessens the possibility of deviations having to do with temperature of the environment, essentially reducing the variables in the experimental design.[25]
Besides yielding information about the barophilicity of certain amino acids, the experiment also provided valuable insight into the origin of the genetic code and its organizational influences. It was found that most of the amino acids that determined barophilicity were also found to be important in the organization of the genetic code. It was also found that more polar amino acids and smaller amino acids were more likely to be barophilic. Through the comparison of these two archaea, the conclusion was reached that the genetic code was likely structured under high hydrostatic pressure, and that hydrostatic pressure was a more influential factor in determining genetic code than temperature.[25]
Pyrococcus furiosus was originally isolatedanaerobically from geothermal marine sediments with temperatures between 90 °C (194 °F) and 100 °C (212 °F) collected at the beach of Porto Levante,Vulcano Island, Italy. It was first described byKarl Stetter of theUniversity of Regensburg in Germany, and a colleague, Gerhard Fiala.Pyrococcus furiosus actually originated a new genus of archaea with its relatively recent discovery in 1986.[1]
The namePyrococcus means "fireball" inGreek, to refer to the extremophile's round shape and ability to grow in temperatures of around 100 degrees Celsius. The species namefuriosus means 'rushing' inLatin, and refers to the extremophile's doubling time and rapid swimming.[1]
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