By Lena VOROBYEVA, Dr. Sc. (Biol.), M.V. Lomonosov Moscow State University

A most significant event in 20th-century biology occurred twenty-six years ago as a new form of life was discovered. These are Archaeota, the organisms remarkable for a peculiar, hitherto unknown mode of existence. Side by side with the domains of eukaryotes and prokaryotes, they constitute what we might call a third domain of life...

Ancient philosophers pointed to the dichotomy (dual, twofold nature) of the living world composed that it was of the plant and animal kingdoms. With the discovery of microorganisms by the Dutch natural scientist Antony van Leewenhoek in 1683 (which he called "little animals") the animal kingdom came to be divided into two essential parts- the micro- and the macro-organisms. And with the appearance of electron microscopy in the 1950s microbiologists, who could now study cell structures, divided all organisms into eukaryotes (with a discrete cell nucleus) and prokaryotes (having no cell nucleus). Accordingly, two types of ribosomes, eukaryotic and prokaryotic, were thought to correspond to these two levels of cell organization. But one had no inkling of two distinct forms of prokaryotic life!

The discovery of a new domain of life by a research team of Illinois University under Carl Woese in 1977 opened a new page in biology. This event is compared to the discovery of Australia (in 1606), a continent heretofore unknown to Europeans who were stupefied to see the kangaroo and eucalyptus trees for the first time there.

Archaeota hold a key to our understanding of the origins of primitive life, of the eukaryotic cell in particular. They enable a better insight into the evolution of metabolism, photosynthesis and information processes. The development of our planet is closely related to the existence of prokaryotes-in fact, the microbes account for more than 90 percent of the philogenetic*, metabolic, molecular and ecological diversity of the earth. The discovery of Archaeota increases this diversity manifold. They make up a significant part of the bio-mass and are found everywhere, both in extreme conditions (in saline bodies of water, alkaline lakes, and in geothermal waters deep within crustal fissures and fresh-water lakes, soil, marine sediments, in the coastal waters of the Antarctic... As the chief component of the oceanic picoplankton (tiny organisms of 0.2 to 2 μm suspended in water), these weights are playing a major part in the world ecosystem.

Unfortunately we know too little about them yet. Dr. Thomas Gold of the United States estimates their underground biomass to be equivalent to the sum total of land surface and water life. Subterranean Archaeota perform exotic chemical reactions,

* With reference to phylogeny, the historical development of organisms, or the evolution of the organic world. -Ed.

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and their very existence is intimately related to geochemistry. Inhaling sulfur or iron, Archaeota oxidize molecular hydrogen and utilize carbon dioxide as their only source of carbon.

Now, the natural question: why did the Archaeota gain the status of life's third domain as late as that? Biologists had been studying them for quite some time without suspecting they had actually been involved with bacteria. How come? The point is that they could discern a novel kingdom of life in Archaeota only after the genome sequencing methods- i.e. determining the DNA and RNA nucleotide sequence-had been developed. Way back in 1965 Linus Pauling (physicist and chemist, Nobel prize, 1954; foreign member of the USSR Academy of Sciences) and Emil Zuckerkandal advanced the hypothesis that some macromolecules (proteins, nucleic acids) could act as evolutionary "chronometers". Say, proteins working like timepieces: changes of the amino acid sequence in them proceed at a steady rate and thus reflect the rate of slow changes in DNA. Such "timepieces" ticking within a genotype may indicate the history and degree of relatedness among organisms. Given the longstanding process of protein synthesis in which ribosomes are implicated, the ribosomal RNA (rRNA) molecule appears to be an excellent object illustrative of relatedness among organisms; Carl Woese and his team hit the nail on the head by choosing this very "chronometer".

Ribosomes are affected but insignificantly by the environment, their evolution is sluggish, and a large number of their copies within DNA "dilute" its fragments transformed into a cell at random. Since ribosomes are abundant in any organism, rRNA can be isolated without preliminary cloning (multiplication). True, what with the polimerase chain reaction method now in wide use (which makes it possible to increase the number of DNA fragments 106 - 109 over within a short time), microbiologists would rather take DNA molecules which we may describe as a "keyboard of life". Many of the new and not yet cultivated Archaeota were discovered through DNA sequencing from only a few cells available in a community under study.

C. Woese used subunit 16S of RNA isolated from a large number of pro-and eukaryotes. Having studied the sequencing results, he came to the astounding conclusion: homologies and differences in the base sequence of prokaryotes happened to be of the same order as those in bacteria and eukaryotic cells. Thereupon it was suggested to divide the living world into three major groups (or three primary kingdoms): bacteria and eukaryotic organelles (mitochondria, chloroplasts); eukaryotes (rather, their cytoplasmic components) and archaebacteria, now known as Archaeota. These were accorded a domain status, alongside with the two domains of bacteria and eukaryotes.

In turn, the domain of Archaeota breaks down into three kingdoms. The first one, Euryarchaeota, comprises extreme halophils (living under high-salinity conditions), thermoplasms (living in abandoned coal pits), certain hyperthermophils (proliferating at 45 - 70o C), methanogens (the only methane-releasing organisms on earth) and microorganisms obtaining energy through the reduction of sulfur, sulfates and other chemical compounds. The second kingdom, Crenarchaeota, is largely represented by hyperthermophils and strict (obligatory) anaerobes inhabiting ocean depths under high pressure or else cold water and soil. The third, Korarchaeota, as found in the hot, iron-rich wellspring of Yellowstone National Park in the United States; these ancient, phylogenetic creatures belong to uncultivated organisms not studied well enough as yet. And the third kingdom of Archaeota, the Nanoarchaeota, is represented by dwarfish (nano) species; they were discovered in 2002 by the German biologist Karl Stetter who isolated them from a deep-water hot spring in the Atlantic Ocean. Nanoarcheota have a genome whose size is at the limit of existence of an independent organism; actually, they are not capable of independent life as obligatory (strict) symbionts (living in cohabitation with some other archaeon).

In their size and appearance Archaeota look like bacteria, though some may have rather bizarre forms of triangles, squares, glass shivers and even hockey sticks. Some strains form long threads composed of 100 cells and more, enclosed in a sheath. Such irregularity of form may be due to the absence of a rigid cell wall.

By now as many as 16 genomes of Archaeota have been studied and described. Archaeota and eukaryotes have many information processes (transcription, translation, DNA metabolism) in common, while a significant part of catabolic (dissimilation) and anabolic (assimilation) reactions proceed like in bacteria. The same is true of "operator genes" (operons). Though composed of different molecules and being different in design, they perform the same functions. But there are also essential differences. For instance, murein- or rather, its component, the muramic acid-was considered to be part of bacterial cell walls as a molecular marker. Archaeota have nothing of the kind. On the other hand, they exhibit great diversity in cell membranes some of which resemble closely eukaryotic tissues (chon-droitin, mucin-protecting gastic epithelial cells); many are covered with tiny beads of the S-layer, which is of glycoproteic nature; and there are also bare cells like those in thermoplasms. Only a few methanogenic Archaeota have a rigid cell wall made of pseudomurein, i.e. not containing muramic acid and D-aminoacids, and devoid of other chemical intermolecular bonds. Therefore antibiotics (penicillin or vancomycin) that kill bacterial cells do not inhibit the growth of Archaeota. Even their motility organs, the flagella, are synthesized differently and are composed of proteins other than bacterial ones (that is containing along with flagellin several other proteins); and they are formed with the participation of the

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leader peptide* and shaperons which hinder protein aggregation. These organisms are slower swimmers than bacteria, especially in a viscous medium. They need not hurry for lack of competitors in harsh habitation media.

Furthermore, Archaeota have different cell membrane lipids and cell membranes than do bacteria and eukaryotes. The former contain non-saponifying esters of glycerol**, ester and polar lipids, isoprenoid carbohydrates, and alkyl benzenes***. They may also contain glycerol esters bound to molecules of isoprenoid alcohol-phytanil, remarkable for high heat resistance. Extreme hyper-thermophilic Archaeota possess other adaptations for life in hot spots: their phytanil chains incorporate five-membered (pentatomic) rings, their number increasing with a temperature rise in the ambient environment.

In eukaryotes and bacteria, however, lipids have an absolutely different structure as represented by glycerol with ester bonds to chains of un-branched fatty acids. Surfactants (phytanil, diphytanil, alkyl benzenes) of Archaeota occur in shale, oil fractions and sedimentary rock. Archaeal lipids resemble native hydrocarbons that predominated in the geologically remote past; possibly, many of such compounds in sedimentary rock or even all of them could have been synthesized by these or related organisms.

The discovery of tetraester lipids in Archaeota has made a revolution in membranology - the very existence of a monolayer membrane structure disagreed with the idea of universal bilayer membrane in all organisms. Archaeota membranes are more resistant to stressors (high temperature, acidity, alkalinity).

As to ribosomes, in Archaeota they have the same dimensions as in bacteria, though in hyperthermophils these particles are more rich in proteins, many of them homologous to ribosomal proteins of enkaryotes. Archaeal RNA polymerase has also affinity with corresponding eukaryotic enzymes (three in number), but it is absolutely unlike bacterial RNA polymerase.

Now let us look into the genome and information processes. The DNA of all organisms, let us recall, is by several orders longer than the cell, and thus it is packed in orderly fashion. This is done by histones**** in eukaryotes, and by small basic proteins in bacteria. But many Euryarchaeota also pack their DNA with the aid of histones, homologous with the two histones in eurkaryotes. This homology extends as far as the protein (initiation) factors implicated in the replication (reproduction) of DNA and transcription (transfer of hereditary information), as confirmed by the following fact: RNA polymerase of eukaryotes recognizes a sequence of DNA nucleotides and initiation (protein) factors involved in transcription, and carries it out after the archaeal DNA template model. The functional homology of the transcription apparatus in both (Archaeota and eukaryotes) has been confirmed in studying protein synthesis by hybrid ribosomes.

A peculiar group of Archaeota, the extreme halophils, populate highly saline bodies of water found usually in hot latitudes. They live in salt-works and in land-locked saline lakes in arid districts, in Russia's south, too. Such microorganisms offer good prospects for practical uses, for instance in facilitating oil extraction: their cells give off exopolysaccharide that increases the viscosity of water injected into wells; halophilic Archaeota can also be used for removing oil slicks and for other utilitarian purposes. Besides, they synthesize bacteriorhodopsin, which is an excellent material for supporting information processes.

* Leader peptide binds to a structural protein and carries it to the flagellum's base. -Auth.

** Isoprene-based compounds characterized by unsaturated bonds. -Auth.

*** Alkyl benzenes contain alkyl substitutes in the benzene ring. -Auth.

**** Histones-basic proteins occurring in complex with DNA (nucleohistone) in cell nuclei; histone are implicated in sustaining and changing the chromosomal structure at different stages of the cell cycle. -Ed.

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A proposed model for the structure and self-assembly of flagella:

a-in bacteria, flagellin subunits pass through the channel and are attached to the upper part of the flagellum;

b-in Archaeota, flagellin subunits linked to chaperons (c) travel toward the cytoplasmic membrane where, digested by preflagellin peptidase (pp), they are incorporated into the base of the nascent flagellum structure;

SL-S-layer;

CM-cytoplasmic membrane;

PS-polar structure.

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Thus, the G.K. Skryabin Institute of Bio-chemistry and Physiology of Micro-organisms (RAS) has suggested innovative photomaterials in which bacteriorhodopsin is added to the film instead of silver. Such films need not to be developed for images which may be erased with blue light so as to record new information again and again. There are plans to make use of bacteriorhodopsin in a new generation of computers enabling dozens of thousands of interlink-ages of elements, something that the semiconductor technology cannot tackle.

Mention should also be made of cold-resistant Archaeota inhabiting the cold waters of the Antarctic at minus 14 - 18C. They settle down on herring fish leaving rusty spots on the scales. Leather and salt crystals are their favorite habitats, too. These tiny creatures keep viable for many years. And they flourish in salt water, for their cell walls, ribosomes, transfer proteins and enzymes function only at high concentrations of sodium and potassium.

Methanogens are pre-eminent among many Archaeota. Microbiologists detected them more than a hundred years ago; and the Russian biologist Vassily Omelyansky even obtained an association of two cultures in 1902-one of them, methane-releasing, was named after him, Methanobacterium omeljanskii.

Methanogens occupy a separate biochemical isle in the sea of prokaryotes as the only microorganisms on earth that generate methane. They utilize a restricted amount of substrates containing a small molecule, and include cofactors* realizing metabolism on the basis of monocarbon compounds-a unique phenomenon absent from the other prokaryotes. These wondrous anaerobes occupy oxygen-free niches and produce methane gaining energy for their growth via graduated reduction of CO2 , or carbon dioxide. They are thought to be the pioneers who settled on the pristine Planet Earth devoid of oxygen, where molecular hydrogen and carbon dioxide were the only primary substrates within reach. Today methanogens inhabit the silt of freshwater and salt lakes, rice paddies, tree trunks, tundra turf, hot water springs, deep underground waters, and the intestine of insects, fish and ruminants. Methanogenic Archaeota include Antarctic and extremely thermophilic species with a temperature maximum 98C, they are present in sea waters and consume mono-, di- and trimethylamines. In 1986, Dr. Tatyana Zhilina (RAS Institute of Microbiology) isolated them from the soda-rich saline lake of Magadi in Kenya.

Methanogens are very sensitive even to minute trace amounts of oxygen, which is a hostile medium to them. That is why they occur in symbiosis with other organisms on which they depend for subsistence and protection against the deleterious effect of air. At the same time methanogens

* That is, factors working together, cooperatively, jointly. CF. coenzymes. -Ed.

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also depend on bacteria cleaving polymer molecules and fermenting monomers to CO2 , H2 and acetate. This fermentation reaction proves thermodynamically favorable should the molecular hydrogen be present at the level of 10 pm. For this reason methanogenic microorganisms are believed to be largely responsible for the anaerobic decomposition of organic matter on our planet and for the turnover of elements in the biosphere.

Biological methanogenesis can be used for obtaining additional energy from a renewable substate, in particular, from industrial wastes and garbage. This is important what with the shrinking fossil fuel reserves. We at the Chemical Enzymology Chair of the Department of Chemistry (Moscow State University) have managed to increase 50 to 100 fold the efficiency of methane production from biomass by using biocatalysts. Ground and macerated, such mass is hydrolyzed with the use of most active enzymes capable of cleaving any cellulose materials. If adopted, this technology could, just within 5 to 7 years, yield half as much bio fuel for urban mass transit.

A fairly large group of Archaeota belongs to the kingdom of Crenarchaeota, so-called hyperthermophilis. Discovered in deep-water thermal springs, these thermophils can grow at temperatures up to +113o C. Most of this group were recovered from geo- and hydrothermal springs, hot soils, and sulfur-rich muds (where they metabolize sulfur); these microorganisms were first detected at a depth of 2.5 km off the Galapago Islands.*

Upon sedimentation water-insoluble sulfides of iron, zinc and gypsum form tall "flues" from which hot water gushes up. It contains significant amounts of manganese ions, molecular hydrogen, carbon monoxide and occasionally, ammonium ions, dissolved oxygen, carbon dioxide and bicarbonate. Continental thermal springs have a similar composition, though they are often acidic because of the presence of sulfuric acid. Highly thermophilic, nonacidophilic and barophilic Archaeota were found in deep thermal springs with chemically neutral waters: the pressure on the sea and ocean floor is hundreds of times as high as the atmospheric pressure.

Research teams of the RAS Institute of Microbiology under Academician Georgi Zavarzin and Professor Yelizaveta Bonch-Osmolovskaya have made a major contribution to the microbiology of deep-water springs. They have found new strains in the water pools of the Valley of Geysers, in the cauldrons of Golovnin and Uzon (Kamchatka), and on the Kunashir Island** - that is, in the volcanic areas of Russia's Far East. Archaeota are also found as deep as 3 km in underground water at t=100o C, their metabolism being closely related to the transformation of substances of geochemical origin: sulfur, metal-containing salts, sulfide minerals, carbon dioxide, molecular hydrogen - i.e. elements and compounds present at the earliest formative stage of our planet. Incipient life underground could avoid the cosmological impacts that sterilized the surface of the globe. That is why the microorganisms of such interest to us half-open the window on those distant times of the earliest evolution of our planet.

... Archaeota, as we have said, account for a significant portion of the global biomass. Given their peculiar biochemical characteristics, these microorganisms may possibly occur on other planets as well. Unfortunately we know but little about their physiology, about their contribution to the sea and dry land ecosystems, and their potential for practical use. And thus we are in for even new striking discoveries.

* See: A. Lisitsyn, A. Sagalevich, "Breakthrough Discovery in the Ocean", Science in Russia, No. 1, 2001. -Ed.

** See: A. Ivanov, "Southern Kuriles: Life Challenged by Odds", Science in Russia, No. 5, 1999. -Ed.

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