Pyrococcus abyssi: Difference between revisions

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5 open reading frames encoding for membrane proteins of P. abyssi were cloned and expressed in a host, Pichia pastoris. A c-myc epitote and 6 His codons were tagged to the 3' end of these genes for immunodetection and purification. Experiments were conducted in Erlenmeyer flasks for the purpose of obtaining a large quantity for purification and structural work of the membrane proteins.
5 open reading frames encoding for membrane proteins of P. abyssi were cloned and expressed in a host, Pichia pastoris. A c-myc epitote and 6 His codons were tagged to the 3' end of these genes for immunodetection and purification. Experiments were conducted in Erlenmeyer flasks for the purpose of obtaining a large quantity for purification and structural work of the membrane proteins (6).


==References==
==References==

Revision as of 00:44, 5 June 2007

A Microbial Biorealm page on the genus Pyrococcus abyssi

Classification

Higher order taxa

Archaea; Euryarchaeota; Thermococci; Thermococcales; Thermococcaceae; Pyrococcus

Species

NCBI: Taxonomy

Pyrococcus abyssi; 272844

Description and significance

Because of their unique ability to survive in an environment of extreme temperatures and pressures, the genome of P.abyssi has been completely sequenced. Since not much has been studied on hyperthermophiles, especially in the pyroccocus species, P. abyssi has been used as a model organism in the laboratories. Hopefully through the study of P. abyssi, a better understanding of the mechanisms for hyperthermophiles can be better understood and that greater genetic tools can be achieved (1).

Genome structure

The genome of P. abyssi is circular and contains 1.76Mb. It has a GC content of 44%. The genome content has not been explored as of yet. Most of the functionality that is known of the P. abyssi genome has come from the comparison to similiar genomes with known functions through BLAST. The comparison of the genome's of P. abyssi and P. horikoshii revealed that archaea's terminus of replication is a major region for replication, similar to that of bacteria. Comparison to another species of pyrococcus, P. furiosus, reveals an "identification of DNA reorganization linked to IS-like elements and DNA integration within tRNA genes" (1). Besides similiarities to these 2 other pyrococcus species, P. abyssi also contains genes which are crenarchaeal and bacterial like. However, these genes are mainly for the use of transport.

P. abyssi has a plasmid, pGT5, which replicates via a rolling circle mechanism. The plasmids of hyperthermophiles have not been studied extensively as some of the other archaea's due to the limited number of organisms that can survive in these extreme conditions. The importance of the study of these organism's genomes and plasmids is to understand their mechanisms of DNA replication, repair, and recombination, especially since it is so different from our own(3).

Cell structure and metabolism

This organism has a Gram-positive cell membrane (1). P. abyssi breaks down peptides and sugars in order to use ATP as an energy source. Instead of using NADH as an electron carrier, P. abyssi utilizes ferrodoxin. Nonetheless, ferrodoxin functions in a similar manner to NADH when it is reoxidized, contributing to a electrochemical gradient which is needed for ATP synthesis. An ATPase is needed for pumping ions across the membrane, yet it is unknown whether this ATPase is utilized for ATP hydrolysis or ATP sythesis. It is not known whether hydrogen or sodium ions are pumped across the membrane to create the membrane potential.(1) In addition, the P. abyssi genome encodes several import and export systems for the transportation of ions across the cell membrane. A sodium/hydrogen antiport is used to maintain a pH of 7 in the cytoplasm and to generate a proton motive force. P. abyssi also encodes for a system where phosphate can be shuttled into and out of the cell via the Pst and Pit systems. The Pit system specifically in P. abyssi encodes for an alkaline phosphatase and 2 unknown proteins which are related to apurinic/apyrimidinic endonucleases (1). The reason for theses extra encoded proteins are still unclear.

The growth of this organism in vitro requires the presence of starch, maltose and pyruvate and not carbohydrates, alcohols, organic acids and amino acids. Also, unlike our own metabolism, P. abyssi does not have a complete citric acid cycle. The incompletion of the TCA cycle occurs when "phosphoenolpyruvate (PEP) and oxaloacetate [is interconverted] by PEP carboxylase and PEP carboxykinase" (1).

Ecology

Pyrococcus abyssi is found near deep sea hyperthermal vents off of the Southwest region of the Pacific Ocean. This organism is classified as a hyperthermophile since it's environment can reach temperatures as high as 102C and pressures can be up to 200 atmospheres. This organism was collected in the smoker material and sea water surrounding these hyperthermal vents. The genome of P. abyssi contains some genes belonging to other archaeas and bacteria's living in the same environment, suggesting gene transfer between these neighboring organisms.

Pathology

This organism is not known to be pathogenic to humans, plants or animals although studies have shown that it possesses a virus-like particle, PAV1. This particle is virus-like since it can reside in a host cell and cause the host cell to continuously release it into the environment. PAV1 resides stably inside of it's host cell without any prophage activity. The infectivity of this particle has not yet been studied due to a lack of a better plaque assay(2).

Application to Biotechnology

Current Research

Cdc6/Orc1 was found to bind onto a 5 kb region in the P. abyssi sequence with great specificity. Also it was found that Cdc6/Orc1 binds to the oriC region therefore the origin of DNA replication (OriC) site must be present within this 5kb region. These conclusions were drawn through the analysis of the results obtained from chromatin immunoprecipitation coupled with hybridization on a whole genome microarray (4).


The PCNA (proliferating cell nuclear antigen) loading onto primed DNA in P. abyssi was enhanced in the presence of replication factor C and B DNA polymerase (poly B). However, in the presence of poly B, on an RNA primed DNA template, the "PCNA/Pol B complex is destabilized in the presence of dNTPs, allowing the family D DNA polymerase (Pol D) to perform RNA-primed DNA synthesis" in the leading strand (5).


5 open reading frames encoding for membrane proteins of P. abyssi were cloned and expressed in a host, Pichia pastoris. A c-myc epitote and 6 His codons were tagged to the 3' end of these genes for immunodetection and purification. Experiments were conducted in Erlenmeyer flasks for the purpose of obtaining a large quantity for purification and structural work of the membrane proteins (6).

References

1.Georges N. Cohen, Valérie Barbe, Didier Flament, Michael Galperin, Roland Heilig, Odile Lecompte, Olivier Poch, Daniel Prieur, Joël Quérellou, Raymond Ripp, Jean-Claude Thierry, John Van der Oost, Jean Weissenbach, Yvan Zivanovic, Patrick Forterre (2003) An integrated analysis of the genome of the hyperthermophilic archaeon Pyrococcus abyssi Molecular Microbiology 47 (6), 1495–1512.


2.C. Geslin,* M. Le Romancer, G. Erauso, M. Gaillard, G. Perrot, and D. Prieur. 2003. PAV1, the First Virus-Like Particle Isolated from a Hyperthermophilic Euryarchaeote, “Pyrococcus abyssi”.J Bacteriol. 2003 July; 185(13): 3888–3894.


3.G Erauso, S Marsin, N Benbouzid-Rollet, M F Baucher, T Barbeyron, Y Zivanovic, D Prieur, and P Forterre. "Sequence of plasmid pGT5 from the archaeon Pyrococcus abyssi: evidence for rolling-circle replication in a hyperthermophile". J Bacteriol. 1996 June; 178(11): 3232–3237.


4. Fujihiko Matsunaga1,2, Annie Glatigny3, Marie-Hélène Mucchielli-Giorgi3, Nicolas Agier3, Hervé Delacroix3, Laetitia Marisa3, Patrice Durosay3, Yoshizumi Ishino2, Lawrence Aggerbeck3 and Patrick Forterre1. Genomewide and biochemical analyses of DNA-binding activity of Cdc6/Orc1 and Mcm proteins in Pyrococcus sp.Nucleic Acids Research, doi:10.1093/nar/gkm212.


5. Rouillon C, Henneke G, Flament D, Querellou J, Raffin JP. "DNA Polymerase Switching on Homotrimeric PCNA at the Replication Fork of the Euryarchaea Pyrococcus abyssi". J Mol Biol. 2007 Jun 1;369(2):343-55. Epub 2007 Mar 24.


6. Labarre C, van Tilbeurgh H, Blondeau K. "Pichia pastoris is a valuable host for the expression of genes encoding membrane proteins from the hyperthermophilic Archeon Pyrococcus abyssi". Extremophiles. 2007 Mar;11(2):403-13. Epub 2006 Nov 8.


7..http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=143


Edited by Stephine Chow, a student of Rachel Larsen and Kit Pogliano