Pyrococcus furiosus

A Microbial Biorealm page on the genus Pyrococcus furiosus

Classification

Higher order taxa

Archaea; Euryarchaeota; Thermococci; Thermococcales; Thermococcaceae; Pyrococcus

Species

Pyrococcus furiosus

Description and significance

Pyrococcus furiosus is an aquatic anaerobe hyperthermophiles archaeon first isolated in a hydrothermal vent near Vulcano Island, Italy. Its optimal growth temperature is 100 0C, so its enzymes are extremely thermo-stable. It is one of the first hyperthermophiles to be studied extensively by scientists, and it was found that its enzymes and proteins are highly resistant to heat shock, and radiation (6). It is also notable that some of its enzymes are tungsten dependent, a very rare element to be found in biological system (5, 18). Moreover, it is unique among its kind in that it can use a wide range of compounds as carbon source, such as peptides, and carbohydrates (14). And unlike other hyperthermophiles, it does not need elemental sulfur for growth (7).

Genome structure

P. furiosus is a single circular chromosome organism. Its genome size is approximately 1.9Mb, with 40.8% of G-C content, and 2,065 open reading frames encoding proteins, 470 operons (4). Of this 2,065 ORFs, 6% (130 ORFs) have been found to be unique to P. furiosus (1, 10). Experimental evidence has shown that it has at least two stable shuttle vectors, and some Insertion Sequence element, suggesting that it has mobile DNA element. And, at least 100 ORFs have been acquired through lateral gene transfer (12).

Cell structure and metabolism

Cell Structure

P. furiosus has flagella that are attached to one pole of the cell. It is composed of mainly one type of glycoprotein similar to bacterial flagellin, but differs in other aspects from bacterial flagella. While bacterial flagella are hollow tube of a single flagellin growing from the tip, archeal flagella are form from many flagellin, and it’s been argued that it might grow from the root (yet to be proven). These flagella are also has very unique function in addition to motility. In about 5% of the cell during stationary phase, the flagella form cable like structure and allow cell to cell connection, a function very much similar to sex pilus in many bacteria. And, it also allows P. furiosus cells to attach itself to a solid surface; along with connection to other cells, P. furiosus can live in a community that’s similar to that of a biofilm of bacteria (12).

Metabolism

Living in such an extreme environment, P. furiosus also has other remarkable mechanism to protect and proliferate itself. In a more general study of hyperthermophiles, it has been found that their enzymes are generally more rigid in structure to prevent from environmental adverse effect (23). Hyperthermophiles also have different proteins properties due to adaptation of their environment. It was found that their proteins are generally denser, having a shorter surface loop length, and solvent exposed surface area (24). In yet another study, it was speculated that the pressure in its environment may help to stabilize its enzymes, raising its resistance against thermal inactivation (25). In addition to this, hyperthermophiles also has a DNA binding protein that helps to protect it from hydrolytic DNA backbone damage (29). P. furiosus was exposed to gamma radiation in a study, and the hydroxyl radical the radiation cause from radiolysis of water did extensive damage to the DNA backbone. But upon exposure, the level of the radA gene, whose protein function to repair DNA damage, is induce to express at high level (6). Moreover, it has ATP dependent chaperonin activity, also known as thermosomes, to help its proteins from thermal inactivation (8). Since it is a hyperthermophiles, it is actually crippling to these organism to be at the lower to moderate range of temperature. When the temperate of a culture of P. furiosus is dropped from 95 0C to 72 0C, there was a 5 hours lag in its growth phase. During this lag phase, it is adjusting its metabolic processes to the cold environment by halting unnecessary metabolic reaction, and enhances transcription of some enzymes needed to maintain viability. During the later stage of this cold shock response, it is adjusting to the environment, and metabolic processes that were initially halted are now returning back to the pre shock level (15). P. furiosus does not have genes to deal with heat shock like other organisms, so how it manages to survive at such extreme temperature is a wonder. In the present of extreme heat, it does induce the formation of appropriate solutes that help to stabilize the cellular proteins against denaturation (16). P. furiosus is very unique among its genus, for it can use both peptide and other carbohydrate as its carbon source. When growing on peptide, P. furiosus needs the presence of elemental sulfur. The exact mechanism of peptide metabolism and the specific role of sulfur in it has yet been uncovered, but it is concluded that it play a role in peptide metabolism (22). It can also use a wide range of carbohydrates, notable are the beta linkage glucose polymers (such as cellubiose, chitin, and laminarin) metabolism. Cellubiose, along with other beta linkage polymers, are taken into the cell by the ABC transport system, and are hydrolyze by one of the 5 enzymes coding for amylase-properties proteins working on beta linkage polymers (13, 19). It also has alpha glucosidase to degrade alpha linkage sugars like maltose (27). P. furiosus has a different glycolytic pathway, using 3 tungsten dependent enzymes, and bypasses the steps that produce NAD(P)H in the better known glycolytic pathway of eukaryotes (5), passing the electron to ferredoxins, which then passes through a hydrogenase to H2. There are four ORFs for hydrogenases in P. furiosus, each having different affinity for H2, the amount of ferredoxin determines the rate of activity for these hydrogenases (3). The ferredoxin in P. furiosus is quite unique in that it is also very thermostable, and has been confirmed that has a different iron-sulfur cluster than others in its genus (28). Another tungsten-containing protein in P. furiosus is suspected to have a role in aldehyde conversion, but more complete details have not been determined (17). Adding to its unique glycolytic pathway are two enzymes, glucokinase and phosphofructokinases; unlike its eukaryotic counterpart, it is ADP dependent rather than ATP dependent. It is speculated to be so because it helps the cell activity to return to normal faster since its cell has a higher concentration of ADP compare to ATP, since ADP is significantly more stable than ATP (18). Since P. furiosus is an anaerobe, exposure to oxygen can be fatal to it. So to deal the oxygen that it inevitable cannot avoid, it has two NADH oxidases, NOX1 and NOX2, to help it deal with oxidative stress cause by the oxygen in the environment. NOX1 catalyzes the oxidation of NADH to both H2O2 and H2O (2). It is unique because it hasn’t been found in another organism that one enzyme can catalyze the formation of both products.

Ecology

Looking at the genome of P. furiosus, scientists have found a spherical protein that’s similar to a bacteriophage. The similarity is only in structure, and not in sequence. Since a virus can enter a host’s genome and remain there as part of the genome to be replicated, this possibly could have been the cause for this spherical viral like protein. But it is suggestive of a common virus ancestor, which then delineate to affect all three domain of life. (9). In its genome, P. furiosus has up to 28 composite transposons, allowing for the DNA to be mobilized to other chromosomes, allowing genetic exchanges in this vent that may have lead to the divergence of other species in the vent (14). It’s been an old belief that genetic information was first encoded in RNA, it was only by the activity of ribonucleotide reductase (RNR) that DNA had evolved. Looking at the RNR in P. furiosus and its similarity to other RNRs, it is suggested that all RNRs came from a common ancestor (26).

Pathology

P. furiosus is not pathogenic.

Application to Biotechnology

P. furiosus have contributed greatly to biotechnology, and potentially will have many more useful contributions in the future as well. Because of the highly thermostable property of the enzymes of P. furiosus, its polymerase, known as Pfu, is widely used for Polymerase Chain Reaction. In addition to this, mutant in the polymerase of the P. furiosus reduces its proof reading capability and causes more error during the replication process, this is helpful when scientist are trying to study the effect of mutations on the function of a gene, mutagenesis (30). Another mutation in the polymerase enhances its affinity for dideoxy-nucleotide triphosphate (ddNTP), which is used for DNA sequencing, allowing for a more sufficient rate of sequencing (32). Another important contribution by the P. furiosus is its amylolytic enzymes capabilities. Since P. furiosus is capable of using alpha and beta linkage carbohydrates, isolation of these enzymes are quite helpful to industrial processes (31).

Current Research

1. P. furiosus has a different glycolytic pathway that bypasses the reduction of NAD(P) to give the electron to ferredoxin and allow the formation of H2 using a membrane bound hydrogenase (MBH). Upon addition of elemental sulfur to the media, the rate of production of H2 decreases and H2S increases. This is due to the up regulation of an NADPH elemental sulfur oxidoreductase (NSR); this also involves the up regulation of a membrane bound oxidoreductase (MBX). Without the elemental sulfur, the H2 formed will be the final product of the glycolytic pathway; but since is H2 toxic to cell growth, getting rid of it is prefer. This study focuses on the role of NSR, and how it signals the positive feedback for MBX to replace the MBH and allow for reduction of elemental sulfur (7). 2. Ferritin is a 24 subunits protein that helps sequester free iron, keeping it in a soluble, nontoxic form until it is needed by the cell. This study focuses on the structure of the ferritin in P. furiosus, and how this structure help protect it from inactivation by the heat. It is proposed that the large number of Hydrogen bonding within the monomer of the proteins allow it to retain its structure, this in turn helps protect the whole proteins from thermal denaturation (11). 3. Being in such extreme environment, P. furiosus has to have mechanism that helps to modified itself against the environment stress. This protein is a dimer, and it interacts with the minor groove to recognize the palindrome of the DNA. This study elucidates the structure of a protein that plays as a heat shock regulator for the organism. It blocks the expression of genes that’s already been damage by the heat until the appropriate precaution against heat shock have been taken to allow the gene to be repair and continue on its way (20). 4. It is essential for polymerase to bind to Proliferating Cell Nuclear Antigen (PCNA) and slides along the DNA in the process of replication. It was already proven that PCNA and DNA Polymerase complex is needed for processive DNA synthesis. In this study with P. furisosus replication, it is demonstrated that the PIP box motif at the C-terminal of the polymerase is needed to load the PCNA onto the polymerase for it to continue on with replication (21).

References

Fiala G. and Stetter K.O. (1986). "Pyrococcus furiosus sp. nov. represents a novel genus of marine heterotrophic archaebacteria growing optimally at 100°C". Archives of Microbiology 145: 56–61.

Edited by ChauNhien Nguyen, student of Rachel Larsen and Kit Pogliano