User:Epicfroggz/Pyrococcus furiosus

I removed the Mre-11 bit because 1) it didn't seem that important or special and 2) the reference it pulls from is a yeast article??? Anyways, replaced it with a section on AdhA, which is important to the genetic engineering we are doing in my lab! - Kat

Introduction

Pyrococcus furiosus is a heterotrophic, strictly anaerobic, extremophilic, model species of archaea. It is classified as a hyperthermophile 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). P. furiosus belongs to the Pyrococcus genus, most commonly found in extreme environmental conditions of hydrothermal vents. It is also one of the few prokaryotic organisms that has enzymes containing tungsten, an element rarely found in biological molecules.

INTRO: Pyrococcus furiosus is a heterotrophic, strictly anaerobic, extremophilic, model species of archaea. It is classified as a hyperthermophile 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 the Pyrococcus genus, most commonly found in extreme environmental conditions of hydrothermal vents and is one of the few prokaryotic organisms that has enzymes containing tungsten, an element rarely found in biological molecules.

Pyrococcus furiosus has many potential laboratory and industrial applications due to its unique thermostable properties. P. furiosus is used in the process of DNA amplification by way of PCR (polymerase chain reaction) because of its proofreading activity. Utilizing P. furiosus in PCR DNA amplification instead of the traditionally used Taq DNA polymerase allows for a significantly more accurate process. The thermodynamic stability of P. furiosus' enzymes is useful in the creation of diols for laboratory and industrial purposes. Certain superoxide dismutases found in P. furiosus can be introduced into plants to increase their tolerance environmentally stressful conditions and ultimately their survival.

Uses

Pfu Polymerase ribbon diagram.

Since the enzymes of P. furiosus are extremely thermostable, the DNA polymerase from P. furiosus (also known as Pfu DNA polymerase) can be used in the polymerase chain reaction (PCR) DNA amplification process. The PCR process must use a thermostable DNA polymerase for automated in vitro amplification and originally used Taq DNA polymerase. However, since purified Taq DNA polymerase lacks exonuclease (proofreading) activity, it cannot excise mismatched nucleotides. Researchers discovered in the early 1990's that the Pfu DNA polymerase of P. furiosus does actually possess a requisite 3’-to-5’ exonuclease activity allowing for the removal of errors. Subsequent tests utilizing Pfu DNA polymerase in the PCR process revealed a more than tenfold improvement over the accuracy of using Taq DNA polymerase.

Properties

Pyrococcus furiosus is a strictly anaerobic, heterotrophic, sulfur-reducing archaea originally isolated from heated sediments in Vulcano, 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. It appears as mostly regular cocci—meaning that it is roughly spherical—of 0.8 µm to 1.5 µm diameter with monopolar polytrichous flagellation. Each organism is surrounded by a cellular envelope composed of glycoprotein called an S-layer

Pyrococcus 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 of P. furiosus is very slow, or nonexistent, on amino acids, organic acids, alcohols, and most carbohydrates (including glucose, fructose, lactose, and galactose). The metabolic products of P. furiosus are CO₂ and H₂. The presence of hydrogen severely inhibits its growth and metabolism; this effect can be circumvented, however, by introducing sulfur into the organism's environment. In this case, H₂S can be produced through its metabolic processes, although no energy seems to be derived from this series of reactions, for the purpose of detoxication or energy conservation, not energy production. but this seems to be due to detoxication or energy-conserving process because it does not produce any energy. Interesting to note is that, While many other hyperthermophiles depend on sulfur for growth, P. furiosus does not.

Interconnected flagella adhering to a solid surface.

A glycoprotein notable to archaea species makes up the majority of the composition of P. furiosus flagella. Aside from potentially using them for swimming, these flagella were observed under lab conditions to be used 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 microcolonial biofilm-like structures.

UPDATED section below reflects changes detailed in above section. Additionally, I moved the paragraph about flagella to be the second paragraph so it would flow nicely after the first paragraph that concludes discussing flagella:

Pyrococcus furiosus is a strictly anaerobic, heterotrophic, sulfur-reducing archaea originally isolated from heated sediments in Vulcano, 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 of glycoprotein called an S-layer. It appears as mostly regular cocci—meaning that it is roughly spherical—of 0.8 µm to 1.5 µm diameter with monopolar polytrichous flagellation.

A glycoprotein notable to archaea species makes up the majority of the composition of P. furiosus flagella. 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 microcolonial biofilm-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 (including glucose, fructose, lactose, and galactose). The metabolic products of P. furiosus are CO₂ and H₂. The presence of hydrogen severely inhibits its growth and metabolism; this effect can be circumvented, however, by introducing sulfur 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.

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 driving ATP synthesis. This could be a very early evolutionary precursor of respiratory systems in all higher organisms today.

Genomics

The sequencing of the complete genome of Pyrococcus furiosus was completed in 2001 by scientists at the University of Maryland Biotechnology Institute. The Maryland team found that the genome has 1,908 kilobases, including 2,065 open reading frames (ORFs) that encode proteins. A study performed in 2005 revealed 17 new ORFs specific to Pyrococcus furiosus that were not originally annotated, bringing the number of ORFs up to 2,082.

A lab strain of Pyrococcus furiosus named COM1 is commonly used for its "high plasticity" and ability to take up foreign DNA, owing to its high recombination and transposon activity. It has 1,571 more base pairs than the referenced NCBI genome, and 10 more insertion 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.

Enzymes

Alcohol dehydrogenases

Pyrococcus furiosus possesses several highly thermostable alcohol dehydrogenases (ADHs): the short-chain AdhA, the iron-containing AdhB, the zinc-containing AdhC, and more. Each of these ADHs are NADP-dependent, and serve to replenish NADP⁺ by using the NADPH produced by fermentation 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.

Oxidoreductases

P. furiosus has five unique tungsten-containing oxidoreductases that are part of its NAD(P)H-independent glycolytic pathway. These enzymes function optimally above 90°C. The first to be discovered was aldehyde ferredoxin oxidoreductase, or AOR, which utilizes tungsten, sulfur, and iron to catalyze the oxidation of aldehydes and reduce ferredoxin (this being the electron carrier instead of NAD(P)H). As this was the first, all tungsten-containing oxidoreductases are said to be part of the AOR family. The next enzyme to be discovered was glyceraldehyde-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 in P. furiosus. Another one is formaldehyde ferredoxin oxidoreductase, or FOR, which catalyzes the oxidation of aldehydes into carboxylic acids. This enzyme utilizes four types of cofactors: tungsten, iron, sulfur, and calcium. The next one, 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. The fifth and final oxidoreductase is named WOR5, and it has a broad specificity for aromatic and aliphatic aldehyde species.

An oxidoreductase species in P. furiosus that does not contain tungsten is pyruvate 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. There is also the indolepyruvate ferredoxin oxidoreductase, or IOR, which utilizes iron and sulfur to catalyze the "oxidative decarboxylation of aryl pyruvates."

Discovery

Pyrococcus furiosus was originally isolated anaerobically 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 by Karl Stetter of the University 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]