A Microbial Biorealm page on the genus Methanopyrus kandleri
Higher order taxa
Description and significance
Methanopyrus kandleri is a Gram positive, rod-shaped, anaerobic methanogen that is classified as an archaeon (2,7,8). Although M. kandleri is considered a methanogen and is phylogically classified with them today, it is considered the most divergent methanogen based on its 16S rRNA sequence as well as many other characteristics. It is believed to be so different from its phylogenic neighbors because of the isolation that is created by its niche.
M. kandleri, is one of the most exceptional extremophiles known today. Not only is it a hyperthermophile, but it is also a thriving halophile. It can survive in temperatures up to 110 degrees Celsius which makes it the most temperature resistant species of all the other methanogens (4,6). This temperature resistance is achieved by the use of its many different adaptations in genome, structure, and enzymes. M. kandleri's hyperthermophilic properties are matched by its halophilic tendencies. Intracellular salt content of trianionic cDPG (cyclic 2,3-diphosphoglycerate) and K+ have been measured at concentrations as high as 1.1M and 3M, respectively (3). Its ability to live in these harsh environments is also thought to be credited towards its methanogenic metabolism (4). Some of the unique enzymes that have been molded by the evolutionary process in this microbe have been proven to be very useful in the biotechnological field. Current research has focused not only on discovering new ways of using this species in the lab, but also figuring out how to classify this complex organism.
The entire genome of M. kandleri has been sequenced by Fidelity Systems in Maryland using a customized sequencing method made specifically for this archaeon called direct genomic sequencing. This process has four phases: the skimming shotgun phase, the direct sequencing phase, the gap closure and assembly verification phase, and finally the computational genome analysis (2). This method actually took advantage of a very unique topoisomerase V found only in M. kandleri to help sequence this genome (5). For more on topoisomerase V, see Cell Structure and metabolism or Current Research. By this method, the genome was determined to be a single chromosome that was 1,694,969 base pairs (bp) long. This method proved to work very well since there was only about 1 error per 40 kb (2). The sequence showed very high levels of guanine and cytosine content, which is most likely an adaptation to the extreme environments it lives in (6). Nucleotides 1694501-747 were believed to contain the origin of replication for this chromosome. In this genome, 1,692 protein-coding genes and 39 structural RNA genes were found. These proteins have the highest ratio between negatively and positively charged amino acids and therefore M. kandleri has the lowest isoelectric point in all archaeon (2). M. kandleri is said to have a very high number of orphan genes (5). Although the 16S rRNA and EF-1alpha sequences phylogenically placed M. kandleri relatively far away from the other species of methanogens, the complete sequencing of the genome showed the similarities between the members of this monophyly group (2,6,7). For more on the classification of this organism, see Current Research.
Cell structure and metabolism
M. kandleri is a gram positive archaeon, which means that it has only one cell membrane that is surrounded by a thick cell wall (2). Because M. kandleri is a hyperthermophile as well as a halophile, many structural changes must take place in order to survive. One example of these changes can be seen in the cell membrane of this organism. The cell membranes show an unusual archaeic characteristic of having unsaturated lipids; specifically terpenoid lipids which are primitive lipids thought to be the origin of phytantyl diethers, found in all other archaea (2,5). Although it was believed that only eukaryotes contained proteins called histones that condense DNA, it was recently discovered that methanogens also had a protein that does this. The histone found in M. kandleri, called HMK, differs from those found in both eukaryotes and other methanogens. HMK is twice as long as other methanogenic histones, but is believed to bind DNA similar to those of eukaryotes based on spatial similarities (10). Another structural rarity can be seen in the reliance of enzymes on intracellular salt content. This salt concentration greatly affects activity and thermostability of enzymes; specifically enzymes involved in methanogenic processes. The two enzymes proven to show sensitivity to salt concentrations are formylmethanofuran:tetrahydromethanopterin formyltransferase and N5,N10-methenyltetrahydromethanopterin cyclohydrolase. In order to protect itself from osmolysis due to high intracellular salt concentrations, M. kandleri is surrounded by a pseudomurien sacculus (2,3,8). The enzyme topoisomerase V is the most rare topoisomerase known and is only found in M. kandleri. When first discovered it was believed to be related to topoisomerase I, but when it was closely examined it was determined that they were in a class of their own (2,5,11). Another extremely rare enzyme found in M. kandleri is a two-subunit reverse gyrase (2,12).
M. kandleri is a methanogen which means that it produces methane from dihydrogen and carbon dioxide in its environment. This process takes many steps and is very complex. It is considered a chemolithoautotroph since it does not use a carbon source other than carbon dioxide. It is also a strict anaerobe which means that it does not use oxygen as its final electron acceptor. Because the methanogenic metabolism of this species is not temperature sensitive, it is able to live in high temperature environments. Because of these high temperatures many of the metabolic enzymes have adapted in many ways as discussed above (2,4,8,13).
M. kandleri was found at the bottom of a “black smoker” chimney 2,000 meters deep on the sea floor of the Gulf of California. Here it takes dihydrogen and carbon dioxide and uses it to make methane through its many metabolic pathways. It is able to do this in its environment mainly because of its modified metabolic enzymes, such as formylmethanofuran:tetrahydromethanopterin formyltransferase and N5,N10-methenyltetrahydromethanopterin cyclohydrolase as discussed above. These enzymes are able to active at high temperatures and in high salt concentrations because of the many negatively charged amino acids that they are constructed from. Their ability to carry out metabolic pathways anaerobically also allows them to live in the bottom of the ocean where oxygen is scarce or nonexistent. M. kandleri genome is also stabilized by its high guanine and cytosine content. This is advantages due to the 3 hydrogen bonds that hold these base pairs together (2,7). Although there is no proven relationship between these microbes and the eukaryotes that thrive in the same to habitat, an association may be found in the future.
There are no known diseases caused by M. kandleri in any species.
Application to Biotechnology
There has only been one discovery to how M. kandleri can be used in the biotechnology field. That is the use of DNA topoisomerase V, an enzyme unique only to M. kandleri, in DNA sequencing. It is such a good choice for this application because of its ability to be active at high temperatures and high salt concentrations. It is also able to handle uneven nucleotide compositions and complex repeat structures unlike other topoisomerases. This enzyme was first used in the ThermoFidelase sequencing kit to determine the sequence of M. kandleri itself.
The helix-hairpin-helix (HhH)2 tandem repeats on the C-terminal tail that make up the DNA binding domain of topoisomerase V are also of interest to many scientists. This HhH2 domain is so interesting because it provides both an apurinic and apyrimidic site that can allow this enzyme to nick the DNA at the phosphodiester bond and remove a single nucleotide. This is important because in vitro the main cause of DNA damage is depurination. Although this HhH2 domain can be found on other enzymes, it is almost never found as abundantly as on topoisomerase V. The strategy for utilizing this domain in vitro is to add them to enzymes like Taq and Pfu DNA polymerases in order to increase their effectiveness and thermostability (5,11, 12).
The discovery of the many ways that distinguish topoisomerase V from all other topoisomerases was recent. Because so many have been found, scientists have been working on finding the origin of this unique enzyme. Patrick Forterre of the Biologie Moleculaire of Gene chez les Extremophiles Intitut in Paris, France has been working on determining if this enzyme may have a viral origin. His hypothesis, that topoisomerase V has a viral origin, is based on the fact that M. kandleri has an unusually high proportion of orphan genes. He believes that these orphan genes originated from viruses because of the diversity of viruses in the biosphere and the rarity of the genes in different microbes. If this is true, he proposes that the viral world, just like the microbial world, has the potential to be full of new and useful enzymes (5).
The recent discovery of the first noneukaryotic histones, lead by Richard L. Fahrner at the University of California, Los Angeles, raised many questions about the relationships between eukaryotes and methanogens. Although the histones found in most methanogens differed greatly from those found in eukaryotes, the histones isolated from M. kandleri are thought to be some what of an intermediate between the two. This unique histone is twice the length of all others found in methanogenic species and contains two histone-fold ms with a single HMK chain which differs from both eukaryotes and other methanogens. Based on spatial similarities, this unique histone is thought to bind DNA analogously to eukaryotic histones. The similarities and differences between the three distinct types of histones known today may provide and incite into the origin of life (10).
Because M. kandleri differs from every other known methanogen, there has been ongoing research to try and discover new ways to classify this organism. The most common strategy for classification, the analysis of 16S rRNA sequences, suggests that there are few similarities between M. kandleri and other methanogens. The discovery of a topoisomerase V that shared some qualities with the eukaryotic topoisomerase 1 also distanced this microbe from the other methanogenic species. The next step to determine the phylogeny of M. kandleri was to analyze its EF-lalpha gene, which is one of the best genes known for determining deep divergences. Eukaryotic EF-1alpha contains an 11-amino acid segment while the EF-1alpha molecule found in methanogens and halogens only contains a 4-amino acid segment. When analyzed, the EF-1alpha molecule found in M. kandleri was determined to have the 4-amino acid segment found in methanogens and halo bacteria and not the 11-amino acid sequence found in eukaryotes. The analysis of M. kandleri’s methyl coenzyme M reductase operon was also found to be unique to archael methanogens only. Other operons have been analyzed and almost all have supported the relation between M. kandleri and the other methanogens. Other evidence includes the lack of cysteinyl-tRNA synthase and the unusual seryl-tRNA synthases. Both of which are characteristic on many methanogens. Further research is still being done to determine the exact phylogeny of this species (2,6,7,13).
(1) NCBI: Methanopyrus kandleri, Accessed August 27, 2007, "http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=2320&lvl=3&lin=f&keep=1&srchmode=1&unlock"
(2) Alexei I. Slesarev, Katja V. Mezhevaya, Kira S. Makarova, Nikolai N. Polushin, Olga V. Shcherbinina, Vera V. Shakhova, Galina I. Belova, L. Aravind, Darren A. Natale, Igor B. Rogozin, Roman L. Tatusov, Yuri I. Wolf, Karl O. Stetter, Andrei G. Malykh, Eugene V. Koonin, and Sergei A. Kozyavkin. "The complete genome of hyperthermophile Methanopyrus kandleri AV19 and monophyly of archaeal methanogens," Proc Natl Acad Sci U S A, 2002 April 2; 99(7): 4644–4649.
(3) Shima S, Thauer RK, Ermler U. “Hyperthermophilic and salt-dependent formyltransferase from Methanopyrus kandleri,” Biochem Soc Trans, 2004 Apr; 32(Pt 2): 269-72.
(4) Ricardo Cavicchioli. “Cold-adapted archaea,” Nature Reviews Microbiology 4, May 2006; 331-343 .
(5) Patrick Forterre. "DNA topoisomerase V: a new fold of mysterious origin," Trends in Biotechnology, June 2006; Volume 24, Issue 6, Pages 245-247
(6) Jork Nolling, Amy Elfner, John R. Palmer, Vanessa J. Steigerwald, Todd D. Pihl, James A. Lake, John N. Reeve. "Phylogeny of Methanopyrus Kandleri Based on Methyl Coenzyme M Reductase Operons;" International Journal of Systematic Bacteriology, Oct. 1996; Vol. 46, No.4, p. 1170-1173.
(7) Maria C. Rivera, and James A. Lake. "The Phylogeny of Methanopyrus kandleri," International Journal of Systematic Bacteriology, Jan. 1996; Vol. 46, No. 1, p. 348-351.
(8) Shima S, Herault DA, Berkessel A, Thauer RK. " Activation and thermostabilization effects of cyclic 2,3-diphosphoglycerate on enzymes from the hypothermophilic Methanopyrus kandleri," Arch Microbio., 1998 Nov; 170(6): 469-72.
(9) Krah R, O'Dea MH, Gellert M. "Reverse gyrase from Methanopyrus kandleri. Reconstitution of an active extremozyme from its two recombinant subunits," J Biol Chem., May 23 1997; 272(21):13986-90.
(10) Fahrner RL, Cascio D, Lake JA, Slesarev A. "An ancestral nuclear protein assembly: crystal structure of the Methanopyrus kandleri histone," Protein Sci., Oct. 2001; 10(10):2002-7.
(11) Alexei I. Slesarev, James A. Lake, Karl O. Stetter, Martin Gellert, and Sergei A. Kozyavkin. "Purification and Characterization of DNA Topoisomerase V," The Journal of Biological Chemistry, Feb 4 1994; p. 3295-3303.
(12) Regis Krah, Mary H. O'Dea, and Martin Gellert. "Reverse Gyrase from Methanopyrus kandleri," The American Society for Biochemistry and Molecular Biology, Inc., May 23, 1997; p. 13986-13990.
(13) Beile Gao and Radhey S Gupta. "Phylogenomic analysis of proteins that are distinctive of Archaea and its main subgroups and the origin of methanogenesis," BMC Genomics, March 29 2007.
Edited by Brandon Leonard, a student of Rachel Larsen
Edited by KLB