Methanopyrus Classification Essay

In this essay we will discuss about Archaebacteria. After reading this essay you will learn about:- 1. Origin of Archaebacteria 2. General Characters of Archaebacteria 3. Typical Characters 4. Important Representatives.

Contents:

  1. Essay on the Origin of Archaebacteria
  2. Essay on the General Characters of Archaebacteria
  3. Essay on the Typical Characters of Archaebacteria
  4. Essay on the Important Representatives of Archaebacteria


Essay # 1. Origin of Archaebacteria:

The archaebacterial have existed on Earth longer than any other organism of any type as their fossils have been located in Africa and Australian rocks 3.5 billion years old. They are such microorganisms which are prokaryotic in their cell organization but possess a strikingly different cell-chemistry when compared with the bacteria.

They are genetically as distantly related to bacteria as both are to eukaryotic organisms. This distinction among the archaebacteria, bacteria and karyotes is so profound that archaebacteria have been given the rank of a domain with a completely different status in the history of organisms’ classification.

Archaebacteria have become present-day’s central dogma of microbiological studies because they are being considered as having much significance:

(i) In relation to the origin and evolution of life, and

(ii) In determining the nature of life.

The archaebacteria are the microorganisms that appear to be relics of such a primitive group which transformed initial environment (which was highly anaerobic and warm) by using variety of compounds as source of carbon and energy before the evolution of the complex photosynthetic process.

It is so said because many of the present-day archaebacteria have been found using organic compounds like formate, acetate, methylamine, methanol, etc., and mostly thrive and prosper in such environmental conditions which can, to some extent, be compared with the environment during the early evolution of life (i.e., the prebiotic environment) on earth.

Archaebacteria, therefore, may prove to be a good test material for analysing various biochemical and biophysical events that might have been associated with the earliest life forms and may help tracing the mysteries of the origin and evolution of life.

Recent studies on enriched water-samples collected from submarine hydrothermal vents reveal that these samples contained cells exhibiting blue-green auto fluorescence (a typical feature of methanogenic archaebacteria) as well as they showed active methane-production at 102°C and some samples even at 300°C.

This is one of the most amazing discoveries related to archaebacteria as their ultrathermophilic existence destroys all current concepts on the upper temperature limits of life. The future studies on these microorganisms, therefore, may lead to new informations on the nature of life itself.


Essay # 2.
General Characters of Archaebacteria:

These prokaryotes often occur in extreme aquatic and terrestrial habitats which may be anaerobic, hyper-saline or hyper-thermophilic. Some archaebacteria have been found as symbionts in animal digestive system. Recently, these have also been collected from very low temperature environments and are thought to constitute upto 34% of the prokaryotic biomass in coastal Antarctic surface waters.

Archaebacteria may be spherical, rod-shaped, spiral, Iobed, plate-shaped, irregularly shaped or pleomorphic. Some are unicellular whereas others are filamentous or aggregates. They range in diameter from 0.1 to over 15 µm. Some filamentous archaebacteria even can grow upto 200 µm in length.

Archaebacteria are both Gram-positive and Gram-negative and reproduce by means of binary fission, fragmentation, budding and as yet unknown mechanisms. They may be aerobic, facultatively anaerobic or obligately anaerobic and, nutritionally, range from chemolithotrophs to organotrophs.


Essay # 3.
Typical Characters of Archaebacteria:

Although archaebacteria are prokaryotic and possess most of the basic features of prokaryotes, they have some very typical characters, which are unique to them and are not found in any other group of organisms. These characters are, cell wall structure, plasma membrane structure, RNA-polymerase structure, tRNA nucleotide sequence, novel coenzymes and cofactors, carbon assimilation pathway, and protein synthesis.

Cell Wall Structure:

Some archaebacteria such as Thermo-plasma species lack cell wall while most others do contain cell walls. The cell wall containing archaebacteria are morphologically almost similar to bacteria, the chemistries of the cell walls of the two are strikingly different.

(i) Morphology:

Archaebacterial cell wall may be gram-positive or gram-negative with respect to gram- staining. The wall of many archaebacteria is thick and homogenous resembling those of Gram-positive bacteria and thus stain Gram-positive (Fig. 7.1 A).

The wall of gram-negative archaebacteria lacks the outer membrane and complex peptidoglycan network present in gram-negative bacteria and, instead, they usually possess a surface layer of protein or glycoprotein subunits (Fig. 7. 1B).

(ii) Chemistry:

Peptidoglycan is the main constituent of the cell wall of all bacteria, but it completely lacks in the cell walls of archaebacteria. The latter shows great biochemical diversity; gram-positive archaebacterial cell walls are made up principally of pseudomurein and polysaccharides, gram-negative ones mainly of protein subunits and glycoproteins.

Methanobacterium, Methanobrevibacter, Methanothermus, and Methanopyrus contain pseudomurein [glycans (sugars) and peptides] in their cell walls.

But, the glycan portion of pseudomurein contains N- acetyltalosaminuronic acid (instead of N-acetylmuramic acid of bacterial glycan) and N-acetylglucosamine which are linked to each other by β (1 → 3) glycosidic bonds instead of β (1 → 4) glycosidic bonds of bacterial cell wall (Fig. 7.2) and alternate to form the cell wall backbone.

Lysozyme cannot digest β (1 → 3) glycosidic bonds. Peptides are short amino acid chains attached to N-acetyltalosaminuronic acid (NAT) and these amino acids are L-amino acids. Penicillin is ineffective in infibiting the cell wall peptide bridge formation.

The walls of some gram-positive archaebacteria contain polysaccharides. Methanosarcina contains non-sulfated polysaccharide similar to chondroitin sulfate of animal connective tissue. Halococcus cell wall is composed of sulfated polysaccharide.

The cell walls of Methanococcus, Thermococcus, Methanomicrobium, Methanogenium, etc. are exclusively made up of protein subunits (protein S-layer), whereas the cell wall of Methanospirillium consists of a protein sheath.

Halobacterium cell wall is made up of glycoproteins but also contains negatively charged acidic amino acids which counteract the positive charges of the high Na+ environment. The cell walls of Methanolobus, Sulfolobus, Thermoproteus, Desulfurococcus and Pyrodictium consist of glycoproteins (glycoprotein S-layer).

Plasma Membrane Structure:

The basic molecular architecture of the plasma membrane is the same in all cellular organisms; it contains lipids whose hydrophobic portion is a long, generally, ‘un-branched’ hydrocarbon chain linked to glycerol by ester linkages (—O—C—).

In contrast to plasma membranes of all cellular organisms, those of archaebacteria contain lipids having ‘branched’ hydrocarbon chains linked to glycerol by ether-linkages (—O—) (Fig. 7.3).

This uniqueness of plasma membrane lipids of archaebacteria is considered as one of the key features that distinguishes them from all other cellular organisms. Since many of the archaebacteria live under very high temperature (up to nearly 100°C) and low pH (up to below 2.0) environments, it is advocated that the unusual lipids of their plasma membrane help them survive under such extreme conditions.

The ether-linkages provide stability to them against thermal breakage and the branching of the hydro­carbon chains decrease membrane fluidity; therefore, plasma membranes are stable under high temperature conditions.

Two types of lipid structures are found among the archaebacterial plasma membranes: glycerol diphytanyl diethers and diglycerol dibiphytanyl tetraethers (Fig. 7.4). Usually the diether side chains are 20 carbons in length and the tetraether chains are 40 carbons.

In general, the coccoid cells of methanogenic archaebacteria contain only glycerol diphytanyl diethers, while the rest contain both glycerol diphytanyl diethers and diglycerol dibiphytanyl tetraethers. The halophilic archae­bacteria contain mainly glycerol diphytanyl diethers, while the thermoacidophilic ones contain mainly diglycerol dibiphytanyl tetraethers.

Archaebacterial plasma membranes containing digycerol dibiphytanyl tetraether give an appearance of a monolayer rather than the usual bilayer because lipid chains are long enough to extend from one side of the membrane to the other (Fig. 7.5). This makes these plasma membranes very resistant to extreme environmental conditions of their habitats.

Genome:

The archaebacterial chromosome, like bacteria, are single closed DNA circles. The latter, in some cases, are significantly smaller than the normal bacterial DNA. Thermoplasma acidophilum DNA is about 0.8 x 109 daltons and Methanobacterium thermoautotrophicum DNA is about 1.1 x 109 daltons in comparison to the DNA of Escherichia coli which are about 2.5 x 109 daltons in size.

In archaebacterial genome the G + C content percentage is high (about 21-68%) and only few plasmids have been reported in archaebacterial cytoplasm.

However, National Centre for Biotechnology Information (USA) listed 19 archaebacteria whose genomes had been sequenced by mid-2004. The completely sequenced genome of Methanococcus jannaschii reveals that 56% of its total 1758 genes are uplike those occurring in bacteria and eukaryotes.

RNA Polymerase Structure:

Archaebacteria, like bacteria, possess a single type of RNA polymerases but the latter are complex consisting of upto 14 subunits (3 or 4 large and others small) as against only few subunits in bacteria (c.f. 5 in E. coli). Archaebacteria RNA polymerases are similar to eukaryote RNA polymerase II. However, archaebacterial RNA polymerases are insensitive to drugs rifampin and streptolygidin.

tRNA Nucleotide Sequence:

The archaebacteria lack ribothymine in the common arm (TΨC arm) of transferRNA (tRNA), whereas it is present in most tRNAs in bacteria and eukaryotes; ribothymine is replaced by pseudouridine or L- methylpseudouridine in archaebacteria. Also, they are methionyl-tRNAmet rather than formylmethionyl- tRNAFmet as indicator-tRNA.

Coenzymes and Cofactors:

The methane production mechanism of methanogenic archaebacteria is unique in the microbial world and involves several novel cofactors and coenzymes not found in any other group of microorganisms.

These coenzymes and cofactors identified so far are:

(i) methanofuran (MP),

(ii) methanopterin (MP),

(iii) coenzyme M (CoM),

(iv) coenzyme F420, and

(v) coenzyme F430 (Fig. 7.6).

Carbohydrate Metabolism:

(i) CO2 Assimilation:

Most of the methanogens and extreme thermophiles enjoy autotrophy, and CO2 fixation takes place in more than one way in them. Thermoproteus and possibly Sulfolobus assimilate carbon dioxide by the reductive tricarboxylic acid cycle (Fig. 7.7A) whereas the methanogens and most of the extreme thermophiles assimilate CO2 by the reductive acetyl-CoA pathway (Fig. 7.7B).

(ii) Carbohydrate Catabolis:

Bacteria and eukaryotes degrade glucose by way of Embden-Meyerhof pathway (glycolysis) whereas the archaebacteria do not use this pathway due to lack of 6-physphofructokinase enzyme in them. Extreme halophiles and thermophiles degrade glucose using a modified form of the Entner-Doudoroff pathway (Fig. 7.8) in which the initial intermediates are not phosphorylated.

Halophiles and extreme thermophiles (e.g., Thermoplasma) degrade glucose using tricarboxylic acid cycle (TCA cycle); no methanogen has yet been reported using complete TCA cycle. Methanogens do not degrade glucose to any significant extent. They, in contrast with glucose degradation, use gluconeogenesis that proceeds by reversal of the Embden-Meyerhof pathway.

(iii)Oxidation of Pyruvate to Acetyl-CoA:

All known archaebacteria have ability to oxidize pyruvate (pyruvic acid) to acetyl-CoA. But, they use the enzyme pyruvate oxidoreductase for this purpose instead of pyruvate dehydrogenase complex used by respiratory bacteria and eukaryotes.

Protein Synthesis:

It has been studied that deviation in protein synthesis both at transcription and translation levels from eubacteria and eukaryotes undoubtedly exist in archaebacteria.


Essay # 4. Important Representatives of Archaebacteria:

Keeping the phylogenetic categorization of archaebacteria under the domain Archaea aside, the archaebacterial representatives can be grouped as under on the basis of their strange metabolic and ecological characteristics: methanogens, extreme halophiles, extreme thermophiles, thermoacidophiles and thermophiles.

(i) Methanogens:

Methanogens are the largest group of archaebacteria characterized by their unique energy metabolism in which they convert fermentation products formed by other anaerobic microorganisms notably CO2, H2, acetate, formate, methylamine and methanol to either methane (CH4) or methane and CO2.

The enzymes used in methanogenesis are very oxygen sensitive, so methanogens are very strict anaerobes (obligate anaerobes) hence produce methane in oxygen-free as well as low redox potential (less than – 333 mv) environment. They are found in all types of anaerobic environments, and are certainly the most prevalent archaebacteria in the ‘moderate’ world.

(a) Representative Genera:

There are many known methanogenic genera, the important ones are Methanobacterium, Methanothermus, Methanococcus, Methanomicrobium, Methanogenium, Methanospirillum, and Methanosarcina. However, a methane producing strain, Methano plasma, has recently been isolated in pure cultures: this strain apparently lacks cell wall.

Methanogens inhabit anaerobic environments rich in organic matter. They have simple nutritional requirements and use large variety of carbon compounds.

Some methanogens can grow as autotrophs and utilize carbon dioxide as a carbon source and hydrogen as an energy source but, they may also utilize formate (a carbon compound) as carbon source. Besides, Methanothrix uses acetate and Methanococcoides utilizes methylamine and methanol. A thermophilic strain of Methanosarcina uses acetate, methylamines, and methanol.

Most known methanogens are mesophilic (requiring optimum temperatures from 21°C to 45°C); some are thermophilic (requiring high temperatures of an optimum between 50°C to 80°C); and so far no psychrophilic (requiring low temperature) methanogens have been isolated.

(b) Methanogenesis (Methane Production):

The methane production mechanism (methanogenesis) is unique in the world of microorganisms and involves several novel group of coenzymes (Coenzyme M, Coenzyme F420 and Coenzyme F430) and cofactors (methanofuran and tetrahydromethanopterin) which are not found in any other group of eubacteria Methane production is an anaerobic environment process and usually occurs at temperatures ranging from 0°C to 100°C.

There are three different metabolic strategies employed by diverse methanogenic archebacteria. Most methanogenens employ the CO2-reducing pathway starting with CO2 or formate.

Others use a Methylotrophic pathway starting with methanol or methylamines (e.g., Methanolobus, Methanoholohium, Methanococcoides). A few methanogens (e.g., Methanosaeta) use an aceticlastic pathway starting with acetate. Methanosarina, however, may use any of the three strategies to produce methane.

CO2-reducing Pathway to Mathane:

The electrons required for the reduction of carbon dioxide to methane can be supplied form H2, formate, CO, alcohols, etc.; it is H2, which is generally used as electron donor during CO2-reducing pathway.

The reduction of CO2 by H2 to produce methane (Fig. 7.9) occurs in the following steps:

(i) CO2 is activated by a methanofuran (MF) and subsequently reduced to the formyl level,

(ii) The formyl group is transferred from methanofuran to methanopterin (MP) and is subsequently dehydrated and reduced to methylene and methyl levels in two separate steps,

(iii) Methaopterin now transfers the methyl group to coenzyme M (CoM),

(iv) The methyl reductase system reduces methyl-CoM to methane (CH4) with the involvement of conenzyme F430 and coenzyme B (CoB), and during this the coenzyme F430 removes the methyl group (CH3) from methyl-CoM in the form of Ni2+-CH3 complex which is reduced by coenzyme B (CoB) generating methane (CH4) and a disulfide complex of CoM and CoB (CoM-S-S-CoB), and

(v) Finally, the disulfide complex (CoM-S-S-CoB) is reduced with H2 to generate free CoM and CoB. It is this reaction during which the energy is conserved in the form of ATP.

(c) Methanogens as Problem-Organisms:

Methanogens can become a problem to ecosystem in future if they are exploited to produce methane. Methane is a greenhouse gas as it absorbs infrared radiation. Evidences are there that the concentrations of methane are continuously rising since many decades and the methane production may significantly aid to the global warming in future.

Recently, it has been found that methanogens oxidize iron into methane and energy. That is, the methanogens growing around buried or submerged iron pipes and other iron objects may bring significant iron corrosion.

Landfill biomass is digested anaerobically to H2, CO2, and acetate which in turn are converted into methane by methanogenesis. Landfills have to be carefully vented to prevent very messy methane explosions because houses near older unvented landfills may explode due to the buildup of methane which may seep through the ground into their basements.

(ii) The Halobacteria: Extreme Halophiles:

The halobacteria, e.g., Halobacterium, Halococcus, etc. are aerobic chemoorganotrophs and salt lovers. They thrive in salt-lakes, tidal pools, salt ponds, brines, salted fish and salted hides. It is considered that they require approximately 17 to 23 per cent NaCl for good growth.

Halobacterium’s cell wall is so dependent on the presence of NaCl that it disintegrates if the NaCl concentration drops to or below 8%. Some of them inhabit deep-sea volcanic vents where the water is maintained boiling due to the extreme pressure of the sea-depth and they may flourish even at 104°C temperature.

The halophiles are poorly flagellated rods (Halobacterium) ranging up to 8 µm in length and immotile cocci (Halococcus). The Halobacterium spp are pleomorphic and can undergo morphological changes in response to variation in the salt content of the environment; a very high salt concentration may cause the cells to become round and ultimately lyse.

Light-Mediated ATP Synthesis: The Role of Bacteriorhodopsin:

The Halobacterium carries on unique version of energy synthesis, namely, light- mediated ATP synthesis (Fig. 7.10) which does not involve photosynthesis. The best studied is the Halobacterium salinarium (H. halobium), which usually traps solar energy without the presence of chlorophyll (thus it is not photosynthesis).

When subjected to low oxygen concentrations and high light intensity, it synthesizes a modified cell membrane, namely, purple membrane, which contains a protein pigment called bacteriorhodopsin. Bacteriorhodopsin was so named due to its structural and functional similarity to the visual pigment of the eye called rhodopsin.

Conjugated to bacteriorhodopsin is a molecule of retinal, a carotenoid-like molecule, which has ability to absorb light and catalyse formation of a proton motive force.

Bacteriorhodopsin absorbs light strongly at about 570 nm light-spectrum. The retinal of bacteriorhodopsin, which normally exists in trans-form, becomes exited and converted to its cis-form following the absorption of light. During this conversion, there is translocation of protons to outside surface of the purple membrane.

The retinal molecule then returns to its trans-form in the dark along with the uptake of a proton from the cytoplasm, thus completing the cycle. As protons accumulate on the outer surface of the membrane, the proton motive force increases until the membrane is sufficiently charged to drive ATP synthesis through action of a proton trans locating ATPase.

(iii) Pyrodictium: Extreme Thermophile:

Pyrodictium is the most extreme example of archaebacteria that grow in extreme thermophilic conditions, particularly the submarine volcanic habitats such as thermal springs and deep sea hydrothermal vents. This archaebacterium has been isolated from geothermally heated sea floors and grow at 82-110°C temperature.

Pyrodictium are irregularly disc-shaped and its cell wall is composed of glycoprotein. It is obligately anaerobic growing chemolithotrophically on H2 with S as electron acceptor or chemoorganotrophically on complex mixture of organic compounds.

How the Pyrodictium, and other extreme thermophiles, tolerate so much heat? In cells of Pyrodictium a heat-shock protein, namely, thermosome, is present. It is considered that this protein functions to keep the other proteins of the cell properly folded and functional and helps cells survive above their maximal growth temperature. For example, the cells of Pyrodictium can survive for one hour at 121°C in the autoclave.

(iv) Sulfolobus, Thermoplasma, Ferroplasma: Thermoacidophiles:

The thermoacidophiles are distinguished by their ability to grow in harsher environments of high temperature and low pH value (acid pH value). They represent a heterogenous group, which can be Sulfolobus, Thermoplasma and Ferroplasma.

Sulfolobus is a gram-negative facultative chemoautotroph. Its cells have an irregular spherical lobed shape. The temperature optimum required is between 70-80°C with a pH optimum of between 2 to 3. Sulfolobus grows in hot acid springs and hot acidic soils all over the world.

The main growth substrate of these is the elemental sulphur. Sulfolobus grows on the surface of droplets or crystals of sulphur and oxidizes it to sulphuric acid which is largely responsible for the acidity of its habitats.

Thermoplasma, like mycoplasmas, lacks cell wall. It is a facultative anaerobe, which utilizes some mono- and disaccharides as source of carbon.

Thermoplasma has been found growing only in the refuse piles from coal mines, which contain residual coal and substantial amounts of iron pyrite (FeS). Oxidation of iron pyrite by some chemoautotrophic bacteria acidifies and heats the pile creating a favourable growth environment for Thermoplasma (temperature 37-65°C; pH 0.5-4.5).

Ferroplasma is a chemolithotrophic relative of Thermoplasma. Like Thermoplasma, it lacks cell wall and enjoys acidophilic life-style, but is not a thermophile as it grows optimally at 35°C in mine tailings containing pyrite (FeS). The latter is used as energy source.

(v) Thermoproteus:

Thermoproteus, a thermophilic archaebacterium, are long thin rods often sharply bent in the middle to produce a V-shaped structure and grow over a temperature range of 78-95°C and a pH range of 2.5-6.5.

Thermoproteus can be identified by the presence of occasional branching of the cells and presence of golf ball-like terminal structures. Their metabolism is strictly respiratory and they use variety of sugars, alcohols, organic acids and carbon monoxide as respiratory substrates.


by Kiri Staiger

Introduction

Methanopyrus kandleri is a species of hyperthermophilic, halotolerant, methanogenic archaeon, originally isolated from sediments associated with undersea, hydrothermal, vent fields. [1] Currently the only species in its genus, M. kandleri is thought to be part of a separate lineage from all other known methanogens, possibly a lineage that diverged extremely early on, close to the time that the kingdoms of life were themselves diverging. [1] Whether or not this is the case is unclear; M. kandleri ’s exact phylogenetic placement, and its significance, have been points of controversy since the microbe’s discovery. [2, 3] Either way, both its unique lineage and its extremophilic nature set M. kandleri apart from its fellows, and it has been the subject of much study and debate.

Classification

Assigning a classification to M. kandleri has been one of the main questions that researchers have debated about the microbe since its discovery in 1991. Although several different classifications have been suggested, the version currently accepted is:

Domain: Archaea
Phylum: Euryarchaeota
Class: Methanopyri
Order: Methanopyrales
Family: Methanopyraceae
Genus: Methanopyrus
Species: kandleri

Species description

Morphology

M. kandleri presents as motile, rod-shaped cells, growing singly or in short chains of generally less than 10 individuals. Individual cells are typically between 8 and 10 microns in length, and half a micron in diameter. Occasional, very short, cells, only about .8 microns in length, are also observed, although whether these micro-cells are merely stunted or an example of intentional cell differentiation (as seen in heterocyst formation in cyanobacteria) is not currently known. Motility in M. kandleri is conferred by flagellar “tufts”, located at cell poles. Cells stain gram-positive; in addition to a psuedomurein inner envelope, cells possess the protein S-layer typical of archaeons. They also exhibit a slime-like layer that coats over the S-layer. It is thought that this slime layer is what allows M. kandleri to form the long, “raft-like” structures seen when the microbe is grown under shaking conditions. [1] It may also assist the microbe in adhering to surfaces. One experiment looking at archaeal bio-film formation found that M. kandleri was highly adherent, able to cling to even the smooth glass surface of a test tube under shaking conditions, where Pyrococcus furiosus (a different, hyperthermophilic, archaeon, whose preferred environmental conditions are comparable to those of M. kandleri) could not. [4] (Figure 2) Certainly, an enhanced ability to stick to one’s environment would not go amiss in M. kandleri ’s home in the walls of underwater thermal vents.

When cultured under less active conditions, M. kandleri may form irregularly shaped colonies, about 1 mm in diameter, that exhibit the blue-green morphology typical of methanogens. [1]


Environmental Preferences

The species was originally isolated from two sites—a “black smoker” hydrothermal vent site in the Guaymas Basin in the Gulf of California, and a shallower hydrothermal vent site at Kolbensey Ridge, off of Iceland. Although the samples were uniformly anaerobic, both sites otherwise exhibit incredible variation, in temperature—between 7° in the cooler sediments and 315° C in the vent-walls themselves—and pressure. The Guaymas Basin site is under 2000 meters of seawater; the Kolbensey Ridge site is much shallower at only around 100 m. [1] M. kandleri does not occupy the entire range of this incredibly diverse environment, but it does exhibit a fairly broad tolerance range, especially in regards to temperature and salinity. The optimum temperature for M. kandleri is 98° C, but the microbe can grow at temperatures as low 84°C and as high as 110°C—ten degrees above the boiling point of water. This extreme hyperthermophilism places M. kandleri among the most heat-tolerant organisms currently known.

Salinity preference for M. kandleri is between 0.2 and 4%, with an optimal salinity of 2%. While not extreme, this does leave M. kandleri as a moderate halophile. The microbe has also been shown to tolerate extremely high levels of internal salinity; like Halobacteria , M. kandleri builds up a high proportion of negatively charged molecules in its proteins and cell structures to counteract the positively charged salts. In the case of M. kandleri, the microbe may tolerate an internal concentration of potassium salts greater than 3M. [1,3]

pH preference is near neutral, between 5.5 and 7; cells grow optimally at a pH of 6.5.

Metabolism

M. kandleri is an obligate, anaerobic, chemolithoautotroph; di-hydrogen gas and carbon dioxide are converted to methane, in a type of metabolism known as methanogenesis. This type of metabolism is found only in archaea, and plays an important role in the carbon cycle, especially in decomposition. Several different types of methanogenesis have been observed. The chemical equation below shows the reactants and involved in the variation of methanogenesis that M. kandleri utilizes.

CO2 + 4 H2 → CH4 + 2H2O

Many methanogens may also use the carbon in organic compounds such as formate or methanol as an electron acceptor, but M. kandleri seems to strictly utilize carbon dioxide in this role. Hydrogen is similarly the sole electron donor—with one exception. Under normal circumstances, the presence of sulfur will only inhibit growth of M. kandleri ; however, sulfur is just similar enough to hydrogen that, under the right circumstances, such as during an exponential growth period where cells are growing and metabolizing rapidly, M. kandleri will mistake it for hydrogen and begin to use it as an electron donor. This is not unusual, per say; many microbes do in fact utilize sulfur in this way to no ill effect. However, one of the byproducts produced by this alternate chemical reaction is hydrogen sulfide (H2S), which appears to be toxic to the microbe, causing high rates of lysis. [1]

Genome type

Methanopyrus kandleri possesses a single, circular chromosome 1,694,969 base pairs in length, containing genes coding for 39 different structural RNAs, and 1,692 different proteins. Of these proteins, only a very few are involved in gene regulation or regulation signaling pathways; fewer than one would predict, even when one takes into consideration that hyperthermophiles as a group are generally less regulated than other microbes. M. kandleri ’s genome also encodes fewer DNA binding proteins, also associated with regulatory activity, than any other known archaeon; this suggests that regulatory activity in M. kandleri is highly reduced, and response pathways may be relatively few. [3]

In addition, M. kandleri codes for an unusually high proportion of negatively charged amino acids in its proteins, which contribute to the microbe’s high proportion of negatively charged residues that, as mentioned previously, is involved in conveying high salt tolerance.

The G+C content is about 60%, which is unusually high compared to most other methanogens, which average only 33% G+C . [1]

Novel proteins and features

A number of novel proteins and other compounds have been isolated from M. kandleri , and are of interest in perhaps shedding light on both its phylogenetic status and its highly thermophilic, halophilic existence. Many hyperthermophilic proteins and enzymes have been described from M. kandleri , and some have found applications in the world of biotechnology. Some of the more interesting discoveries, in terms of either evolutionary significance or possible applications, are outlined in this section.

Novel Psuedomurien Variate

Many archaeons possess a cell wall consisting of pseudomurein. Also called pseudopeptidoglycan, pseudomurein acts much as peptidoglycan does in bacterial cell walls, forming a thick, multi-layered envelope around the cell. Ordinarily, pseudomurein is made up of N-acetylglucosamine and N-acetyltalosaminuronic acid. However, Methanopyrus kandleri substitutes L-orthinine where N-acetylglucosamine ordinarily would go.

Novel Core Lipid

A number of novel lipids have been isolated from M. kandleri. The figure at right shows the structure of one of these lipids, 2,3-di- O-phytanyl-sn-glycerophosphocholine (Fig. 3). In addition, the microbe was found to utilize a novel core lipid. This lipid, an unsaturated, ether-linked lipid identified as 2,3-di-O-geranylgeranyl-sn-glycerol, is of a type of lipids (terpenoid lipids) thought to be a ‘primitive feature’ of many microbes. The presence of this terpenoid lipid may, in combination with other evidence (as detailed later in this article), provide some evidence for the claim that M. kandleri is of an early diverging branch from the Euryarchaeota group. [12]

Novel DNA-binding proteins

Sequence analysis of the M. kandleri genome shows a relative deficiency in this microbe of the helix-turn-helix and Arc-MetJ type DNA binding proteins that are usually found in abundance across both archaeons and bacteria. Instead, M. kandleri appears to utilize (at least) 3 novel DNA binding proteins. All are relatively small molecules, with molecular weights of 7, 10, and 30 kDa. The structure of the 7 kDa binding protein has been determined to be dimeric, consisting of a larger alpha-helical protein associated with a smaller, N-terminal beta-strand. Structural properties, as well as apparent regulatory similarities, suggest that this binding protein may be a relative of the ribbon-helix-helix family of transcription factors that are involved in DNA binding and gene regulation in bacteria, notably Pseudomonas bacteria. [10]

Hetero-dimeric reverse DNA gyrase

Reverse DNA gyrases, which positively supercoil DNA when activated by ATP, have been found to be common across nearly all hyperthermophiles, both archaeons and bacteria. However, the M. kandleri DNA reverse gyrase is the first hetero-dimeric DNA reverse gyrase to be described. The protein consists of two sub-units—a larger (138 kDa) subunit that associates with ATP and hydrolyses it to release energy; and a smaller (43 kDa) sub-unit that binds covalently to DNA as the protein works to create supercoils. The presence of these two sub-units within the gyrase, as opposed to having a single unit protein, is thought to help stabilize the protein at the incredibly high temperatures M. kandleri is subject to. In addition, high temperatures appear to be required for the two sub-units to form the stable heterodimer observed. [9]

Novel and modified RNA nucleosides

M. kandleri shows an unusual and highly diverse pattern of post-transcriptional modifications to tRNA nucleosides—4 entirely novel nucleosides have been isolated from this microbe. Modifications to these nucleosides effect structure, behavior, and stability of the tRNA strands each helps to form. The high degree of modification seen in M. kandleri may have to do with the extreme nature of its environment and the high demands that environment places on its proteins in terms of maintaining stability. [11]

Questions and Controversy- Placing M. kandleri within the evolutionary tree of life

Since its discovery, the placement of M. kandleri within a broader phylogenetic context has been a highly contested avenue of research. At different times, it has been thought to be many different things—a representative of a new class within Euryarchaeota , only very distantly related to its fellow methanogens [1, 2]; alternatively, quite closely related to other methanogens [3]; or, even more alternatively, quite unrelated from just about everything, and possibly a link to finding the much lauded Last Universal Common Ancestor [5]. Which one of these M. kandleri actually is, is still up to debate.

Initial Classification

The researchers who initially isolated the microbe placed it in a separate, deeper-branching, clade from other methanogens based on the following observations:

First, the G+C content of Methanopyrus was much higher than that of other known methanogens. At 60%G+C, Methanopyrus has almost twice the G+C in its genome as other methanogens. Second, the amount of homologous DNA sequences observed between Methanopyrus and other methanogens was, in their words, “insignificant.” Third, lipid analysis of the structures of Methanopyrus revealed a very different set of core lipids than what ought to be a closely related archaeons, Methanococcus . Where Methanococcus ’ core lipids are 85% comprised of cyclic dibisphytanyl tetraethers, Methanopyrus ’ core lipid was 2,3-di-O-phytanyl-glycerol, a very different type of lipid. Fourth, Methanopyrus had a higher halo-tolerance than other Methanobacteriales species. Fifth, Methanopyrus exhibited a novel type of psuedomurein in its cell wall, a variant that substituted L-ornithine for N-acetylglucosamine, and had not been observed previously. Sixth, and perhaps most convincingly, phylogenetic analysis of the microbe’s 16s RNA sequence placed it quite distantly from all known methanogens. In fact, 16s RNA analysis placed it so very distantly from other methanogens that the researchers designated it in a new class—Class Methanopyri, which they had branching off from the Phylum Euryarchaeota significantly earlier than any other known branch. [1]

Support for a separate lineage

In the research since Methanopyrus ’ initial discovery in 1991, there has been some support for this theory. Analysis of M. kandleri ’s 16s RNA sequence suggests that, although a Euryarchaote, Methanopyrus shares a higher degree of relatedness to the chrenarchaeota group that seen in any other Euryarchaeote , which in turn suggests that M. kandleri may have diverged very early on. A 1996 study used a Jukes-Cantor distance projection to approach the problem qualitatively, looking at distances between 16s RNA sequences and regions of the 1-α elongation factor for M. kandleri and proposed relations, including methanogens. The tree generated by this projection again placed Methanopyrus at a deep branch within Euryarchaeota , existing either within or proximal to archaebacteria. [2] (Figure 4) A similar study using the sequences from an operon common across methanogens, coding for methyl coenzyme M reductase, also supported the conclusion that Methanopyrus stands separate from other methanogens. [6]

If this view of Methanopyrus phylogeny is correct and Methanopyrus is of a separate lineage than other methanogens, this would indicate that methanogenesis as a metabolism has arisen at least two separate times. Perhaps more importantly, because many phylogenetic analyses of Methanopyrus place it as a representative of a very deep branching group, some have looked to Methanopyrus as perhaps the most proximal known relative of the Last Universal Common Ancestor, or LUCA. Although previous efforts to locate the LUCA placed it somewhere in the bacterial realm, more recent analyses, incorporating a greater diversity of organisms, especially the previously under-represented archaeons, tell a different story. Phylogenetic distances based on tRNA sequences, which show much lower rates of horizontal transfer than protein coding genes and, as such, are more likely to generate accurate phylogenetic trees over this kind of time-frame, suggest that the LUCA falls somewhere between Euryarchaeota and Crenarchaeota —just about, in fact, where Methanopyrus kandleri seems to fit. [8] If this is the case, than M. kandleri and its genome can tell us a lot about the early divergence of life.

Support for a monophyletic view of M. kandleri and the methanogens

As with any radical claim in science, the proposal of a Methanopyrus-proximal LUCA met with immediate refutal. Opposing this view—opposing, in fact, the entire idea that Methanopyrus is of a different lineage than its fellow methanogens—we have a second set of research. One study used full DNA sequence data and the idea of COGs—Clusters of Orthologous Groups, sets of gene orthologs that are conserved across different phylogenetic groups—to construct a very different phylogeny, that grouped M. kandleri in one lineage with other methanogens. According to this work, about 73% of the proteins coded for in M. kandleri ’s genome are part of COGs shared across a broad spectrum of life; many of the remaining proteins are common across methanogens, which supports the proposal of a single methanogenic lineage. In the trees generated by this COG analysis, M. kandleri is shown as most proximal to either Methanococcus thermoautotrophicum or Methanococcus jannaschii.

The sequence data for these three species—Methanococcus thermoautotrophicum, Methanococcus jannaschii , and Methanopyrus kandleri —is somewhat ambiguous as to who exactly is most related to whom. All three share COGs that are common across methanogens. M. jannaschii and M. thermoautotrophicum share a partial duplication in a recently described operon. The placement and order of genes and operons is more similar between M. thermoautotrophicum and M. kandleri . Depending on what metric we pick, these three species arrange themselves into various relationships. But, despite this ongoing fine-tuning, this research makes a clear case for placing Methanopyrus within a single lineage with the methanogens. (Figure 5) [3]

Further muddying the waters, it has been suggested that many of the phylogenetic analyses that have placed M. kandleri in a separate lineage may be reflective not of an ancient divergence but of an unusually high mutation rate. Hyperthermophilic conditions such as those that M. kandleri thrives in are known to effect the stability of DNA secondary structures, causing hyperthermophilic microorganisms to exhibit higher than normal rates of sequence mutation. So one school of thought would hold that this relative genetic instability would skew any genetic distance analyses of M. kandleri towards predicting a much earlier divergence than actually occurred. On the other hand, mechanisms have been observed in many non-archaeal hyperthermophiles that mitigate wholly or entirely this effect, lowering their mutation rates to something more comparable to non-hyperthermophiles. These mechanisms, or similar mechanisms, may be in effect in M. kandleri-- however, because the DNA repair genes that encode these mechanisms are not well known in archaea, we just don't know at this point whether this is the case. (14) As seems to be often the case with M. kandleri, we are left questioning.

So—what to do with Methanopyrus, in the end? Is it a radically new lineage of methanogen, presenting new insight into the last universal origins of life itself? Is it nothing so bold, just another methanogen? With evidences that seem to point in all directions, this question is not yet resolved, and may not be any time soon. Which evidences do we choose? Do we base our phylogenetic analysis off of 16s RNA? Whole genome analysis? tRNA sequences? Whichever way we do it, we seem to get a different result. The paradox of M. kandleri remains.

References

[6] 1. Margit Kurr, Robert Huber, Helmut Konig, Holger W. Jannasch, Hans Fricke, Antonio Trineone, Jakob K. Kristjansson, and Karl O. Stetter. 1991. Methanopyrus kandleri ,gen. and sp. nov. represents a novel group of hyperthermophilic methanogens, growing at 110° C. Arch Microbiol 156:239—247.

[7] 2. Rivera, M.C., and Lake, J. 1996. The Phylogeny of Methanopyrus kandleri . IJSEM 46: 348-351.

[8] 3. Slesarev AI, Mezhevaya KV, Makarova KS, Polushin NN, Shcherbinina OV, et al. 2002. The complete genome of hyperthermophile Methanopyrus kandleri AV19 and monophyly of archaeal methanogens. Proc Natl Acad Sci U S A 99: 4644–4649.

[9] 4. Simone Schopf, Gerhard Wanner, Reinhard Rachel, and Reinhard Wirth. 2008. An archaeal bi-species biofilm formed by Pyrococcus furiosus and Methanopyrus kandleri . Archives of Microbiology 190: 371-377.

[10] 5. Zhiliang Yu, and Shanxiang Xu. 2011. Search for a Methanopyrus -proximal last universal common ancestor based on comparative-genomic analysis. Annals of Microbiology 61: 397-401.

[11] 6. JORK NOLLING, AMY ELFNER, JOHN R. PALMER, VANESSA J. STEIGERWALD, TODD D. PIHL, JAMES A. LAKE, AND JOHN N. REEVE. 1996. Phylogeny of Methanopyms kandleri Based on Methyl Coenzyme M Reductase Operons. INTERNATIONAL JOURNAL oF SYSTEMATIC BACTERIOLOGY. 46: 1170-1173.

[12] 7. Zhiliang Yu, Ken Takai, Alexei Slesarev, Hong Xue and J. Tze-Fei Wong. 2009. Search for Primitive Methanopyrus Based on Genetic Distance Between Val- and Ile-tRNA Synthetases. Journal of Molecular Evolution 69: 386-394

[13] 8. S. Burggraf, K.O. Stetter, P. Rouviere, C.R. Woese. 1991. Methanopyrus kandleri : An Archaeal Methanogen Unrelated to all Other Known Methanogens. Systematic and Applied Microbiology 14: 346-351.

[14] 9. Regis Krah, Mary H. O’Dea and Martin Gellert. 1997. RECONSTITUTION OF AN ACTIVE EXTREMOZYME FROM ITS TWO RECOMBINANT SUBUNITS. The Journal of Biological Chemistry, 272, 13986-13990.

[15] 10. Nikolai A. Pavlov, Dmitry I. Cherny, Igor V. Nazimov, Alexei I. Slesarev, and Vinod Subramaniam. 2002. Identification, cloning and characterization of a new DNA-binding protein from the hyperthermophilic methanogen Methanopyrus kandleri . Nucl. Acids Res. (2002) 30 (3): 685-694.

[16] 11. Sauerwald, Anselm1; Sitaramaiah, Devarasetty2; McCloskey, James A.2,3; Söll, Dieter1; Crain, Pamela F.. 2005. N6-Acetyladenosine: A new modified nucleoside from Methanopyrus kandleri tRNA 579: 2807-2810.

[17] 12. HAFENBRADL, D; KELLER, M; THIERICKE, R; STETTER, KO. 1993. A NOVEL UNSATURATED ARCHAEAL ETHER CORE LIPID FROM THE HYPERTHERMOPHILE METHANOPYRUS -KANDLERI. SYSTEMATIC AND APPLIED MICROBIOLOGY 16: 165-169.

[18] 13. MASATERU NISHIHARA, HIROYUKI MORII, KOJI MATSUNO, MAMI OHGA, KARL O. STETTER, and YOSUKE KOGA. 2002. Structural analysis by reductive cleavage with LiAlH4 of an allyl ether choline-phospholipid, archaetidylcholine, from the hyperthermophilic methanoarchaeon Methanopyrus kandleri. Archaea 1, 123-131.

[19] 14. Dennis W. Grogan. 1998. Hyperthermophiles and the problem of DNA instability. Molecular Microbiology 28(6):1043–1049.

Figure 1: Micrograph of M. kandleri cells published in the original paper that first described the species. (1) [1]
Figure 2: Micrograph of M. kandleri cells (rods) growing in biofilm with P furiosus (cocci). M. kandleri is unusual in its ability to attach itself to glass under the shaking conditions in which this biofilm forms. Although the furiosus bacteria are unable to adhere to the smooth surface of the glass, they are quick to adhere to M. kandleri, resulting in a two species biofilm, with kandleri usually being layered over by several layers of furiosus cells. (4) [2]
Figure 3: Molecular structure of a novel, choline containing, allyl ether-linked phospholipid isolated from M. kandleri, along with other novel lipids. archaetidylcholine (2,3-di- O-phytanyl-sn-glycerophosphocholine) Figure from Nishihara et al (13)[3]
Figure 4: Proposed phylogenies that would place M. kandleri in a separately diverging group from the other methanogens, and possibly closer to the Eocyte lineage. (2) [4]
Figure 5: Alternatively proposed phylogenies for M. kandleri ("Metka") based on COG-based DNA distance analysis. These phylogenies place Methanopyrus in a single, closely related clade with the other methanogens. (3) [5]

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