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Introduction to the Archaea Life's extremists

The Domain Archaea wasn't recognized as a major domain of life until quite recently. Until the 20th century, most biologists considered all living things to be classifiable as either a plant or an animal. But in the 1950s and 1960s, most biologists came to the realization that this system failed to accomodate the fungi, protists, and bacteria. By the 1970s, a system of Five Kingdoms had come to be accepted as the model by which all living things could be classified. At a more fundamental level, a distinction was made between the prokaryotic bacteria and the four eukaryotic kingdoms (plants, animals, fungi, & protists). The distinction recognizes the common traits that eukaryotic organisms share, such as nuclei, cytoskeletons, and internal membranes. The scientific community was understandably shocked in the late 1970s by the discovery of an entirely new group of organisms -- the Archaea. Dr. Carl Woese and his colleagues at the University of Illinois were studying relationships among the prokaryotes using DNA sequences, and found that there were two distinctly different groups. Those "bacteria" that lived at high temperatures or produced methane clustered together as a group well away from the usual bacteria and the eukaryotes. Because of this vast difference in genetic makeup, Woese proposed that life be divided into three domains: Eukaryota, Eubacteria, and Archaebacteria. He later decided that the term Archaebacteria was a misnomer, and shortened it to Archaea. The three domains are shown in the illustration above at right, which illustrates also that each group is very different from the others. Further work has revealed additional surprises, which you can read about on the other pages of this exhibit. It is true that most archaeans don't look that different from bacteria under the microscope, and that the extreme conditions under which many species live has made them difficult to culture, so their unique place among living organisms long went unrecognized. However, biochemically and genetically, they are as different from bacteria as you are. Although many books and articles still refer to them as "Archaebacteria", that term has been abandoned because they aren't bacteria -- they're Archaea.

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Finding Archaea : The hot springs of Yellowstone National Park, USA, were among the first places Archaea were discovered. At left is Octopus Spring, and at right is Obsidian Pool. Each pool has slightly different mineral content, temperature, salinity, etc., so different pools may contain different communities of archaeans and other microbes. The biologists pictured above are immersing microscope slides in the boiling pool onto which some archaeans might be captured for study.

Archaeans include inhabitants of some of the most extreme environments on the planet. Some live near rift vents in the deep sea at temperatures well over 100 degrees Centigrade. Others live in hot springs (such as the ones pictured above), or in extremely alkaline or acid waters. They have been found thriving inside the digestive tracts of cows, termites, and marine life where they produce methane. They live in the anoxic muds of marshes and at the bottom of the ocean, and even thrive in petroleum deposits deep underground. Some archaeans can survive the dessicating effects of extremely saline waters. One salt-loving group of archaea includes Halobacterium, a well-studied archaean. The lightsensitive pigment bacteriorhodopsin gives Halobacterium its color and provides it with chemical energy. Bacteriorhodopsin has a lovely purple color and it pumps protons to the

outside of the membrane. When these protons flow back, they are used in the synthesis of ATP, which is the energy source of the cell. This protein is chemically very similar to the lightdetecting pigment rhodopsin, found in the vertebrate retina. Archaeans may be the only organisms that can live in extreme habitats such as thermal vents or hypersaline water. They may be extremely abundant in environments that are hostile to all other life forms. However, archaeans are not restricted to extreme environments; new research is showing that archaeans are also quite abundant in the plankton of the open sea. Much is still to be learned about these microbes, but it is clear that the Archaea is a remarkably diverse and successful clade of organisms.

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Archaea: Fossil Record

The search for fossils of Archaea faces a number of problems. First of all, they're very tiny organisms and so will leave microscopic fossils. Any search for fossilized archaeal cells would require a lot of time spent with a microscope and a lot of patience. In fact, there are fossil microbes known from throughout the Precambrian, but here a second problem surfaces -- how do you distinguish fossil archaeans from fossil bacteria? Archaea and Bacteria cells may be of similar sizes and shapes, so the shape of a microbial fossil does not usually help in determining its origin. Instead of physical features, micropaleontologists rely on chemical features. Chemical traces of ancient organisms are called molecular fossils, and include a wide variety of chemical substances. Ideally, a molecular fossil should be a chemical compound that (1) is found in just one group of organisms, (2) is not prone to chemical decay, or (3) decays into predictable and recognizable secondary chemicals.

Not so inhospitable : It used to be unthinkable that life could exist at temperatures near boiling, but some intrepid archaeans thrive under these conditions. Geysers, like the one shown above, are home to such microbes and may help us understand how life existed when the Earth was young.

In the case of the Archaea, there is a very good candidate to preserve as a molecular fossil from the cell membrane. Archeal membranes do not contain the same lipids (oily compounds) that other organisms do; instead, their membranes are formed from isoprene chains. Because these particular isoprene structures are unique to archaeans, and because they are not

as prone to decomposition at high temperatures, they make good markers for the presence of ancient Archaea. Molecular fossils of Archaea in the form of isoprenoid residues were first reported from the Messel oil shale of Germany (Michaelis & Albrecht, 1979). These are Miocene desposits 3

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whose geologic history is well known. Material from the shale was dissolved and analyzed using a combination of chromatography and mass spectrometry. These processes work by separating compounds by weight and other properties, and produce a "chemical fingerprint". The fingerprint of the Messel shale included isoprene compounds identical to those found in some archaeans. Based on the geologic history of the Messel area, thermophiles and halophiles are not likely to have ever lived there, so the most likely culprits to have left these chemical fingerprints behind are archaeal methanogens (methaneproducers). Since their discovery in the Messel shales, isoprene compounds indicative of ancient Archaea have been found in numerous other localities (Hahn & Haug, 1986), including

Mesozoic, Paleozoic, and Precambrian sediments. Their chemical traces have even been found in sediments from the Isua district of west Greenland, the oldest known sediments on Earth at about 3.8 billion years old. This means that the Archaea (and life in general) appeared on Earth within one billion years of the planet's formation, and at a time when conditions were still quite inhospitable for life as we usually think of it. The atmosphere of the young Earth was rich in ammonia and methane, and was probably very hot. Such conditions, while toxic to plants and animals, can be quite cozy for archaeans. Rather than being oddball organisms evolved to survive in unusual conditions, the Archaea may represent remnants of once-thriving communities that dominated the world when it was young.

Archaea: Ecology
Archaeans include inhabitants of some of the most extreme environments on the planet. Some live near rift vents in the deep sea at temperatures well over 100 degrees Centigrade. Others live in hot springs, in extremely alkaline or acid waters, or in extremely saline water. These pictures show an immense bloom of a halophilic ("salt-loving"; dependent on high salt concentrations) archaean species, in a saline pond at a salt works near San Quintin, Baja California Norte, Mexico. This archaean, Halobacterium, also lives in enormous numbers in salt ponds at the south end of San Francisco Bay; interested residents of this area should take the 4

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Dumbarton Bridge for the best views. An interesting fact about Halobacterium is that the red lightsensitive pigment that gives Halobacterium its color, which is a simple photosynthetic system that

provides the archaean with chemical energy, is known as bacteriorhodopsin -- and is chemically very similar to the light-detecting pigment rhodopsin, found in the vertebrate retina.

Salt-lovers : immense bloom of a halophilic ("salt-loving") archaean species at a salt works near San Quentin, Baja California Norte, Mexico. This archaean, Halobacterium, also lives in enormous numbers in salt ponds at the south end of San Francisco Bay; interested residents of this area should take the Dumbarton Bridge for the best views.

Archaea: Systematics
The Archaea constitute one of the three domains into which all known life may be divided. There are two other domains of life. One of these is the Eukaryota, which includes the plants, animals, fungi, and protists. Except for the protists, these organisms have been known and studied since the time of Aristotle, and are the organisms with which you are most likely familiar. The second domain to be discovered was the Bacteria, first observed in the 17th century under the microscope by people such as the 5

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Dutch naturalist Leeuwenhoek.

Antony

van

The tiny size of bacteria made them difficult to study. Early classifications depended on the shape of individuals, the appearance of colonies in laboratory cultures, and other physical characteristics. When biochemistry blossomed as a modern science, chemical characteristics were also used to classify bacterial species, but even this information was not enough to reliably identify and classify the tiny microbes. Reliable and repeatable classification of bacteria was not possible until the late 20th century when molecular biology made it possible to sequence their DNA.

Molecules of DNA are found in the cells of all living things, and store the information cells need to build proteins and other cell components. One of the most important components of cells is the ribosome, a large and complex molecule that converts the DNA message into a chemical product. Most of the chemical composition of a ribosome is RNA, a molecule very similar to DNA, and which has its own sequence of building blocks. With sequencing techniques, a molecular biologist can take apart the building block of RNA one by one and identify each one. The result is the sequence of those building blocks.

A New Domain : In the late 1970s, Dr. Carl Woese (pictured above at left) spearheaded a study of evolutionary relationships among prokaryotes. Instead of physical characters, he relied on RNA sequences to determine how closely related these microbes were. He discovered that the prokaryotes were actually composed of two very different groups -- the Bacteria and a newly recognized group that he called Archaea. Each of these groups is as different from the other as they are from eukaryotes. These three groups are now recognized as three distinct domains of life, as shown above at right.

Because ribosomes are so critically important is the functioning of living things, they are not prone to rapid evolution. A major change in ribosome sequence can render the

ribosome unable to fulfill its duties of building new proteins for the cell. Because of this, we say that the sequence in the ribosomes is conserved -- that it does not change much over 6

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time. This slow rate of molecular evolution made the ribosome sequence a good choice for unlocking the secrets of bacterial evolution. By comparing the slight differences in ribosome sequence among a wide diversity of bacteria, groups of similar sequences could be found and recognized as a related group. In the 1970s, Carl Woese and his colleagues at the University of Illinois at Urbana-Champaign began investigating the sequences of bacteria with the goal of developing a better picture of bacterial relationships. Their findings were published in 1977, and included a big surprise. Not all tiny microbes were closely related. In addition to the bacteria and eukaryote groups in the analysis, there was a third group of methane-producing microbes. These methanogens were already

known to be chemical oddities in the microbial world, since they were killed by oxygen, produced unusual enzymes, and had cell walls different from all known bacteria. The significance of Woese's work was that he showed these bizarre microbes were different at the most fundamental level of their biology. Their RNA sequences were no more like those of the bacteria than like fish or flowers. To recognize this enormous difference, he named the group "Archaebacteria" to distinguish them from the "Eubacteria" (true bacteria). As the true level of separation between these organisms became clear, Woese shortened his original name to Archaea to keep anyone from thinking that archaeans were simply a bacterial group.

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Archaean Phylogeny : The phylogeny of archaeans is based on molecular sequences in their DNA. The analysis of these sequences reveals three distinct groups within the Archaea. The Euryarcheota are probably the best known, including many methane-producers and salt-loving archaeans. Crenarcheota include those species that live at the highest temperatures of any known living things, though a wide variety have recently been discovered growing in soil and water at more moderate temperatures. The Korarcheota are only known from their DNA sequences -- nothing more is known about them yet since they have only recently been discovered.

Since the discovery that methanogens belong to the Archaea and not to the Bacteria, a number of other archaeal groups have been discovered. These include some truly weird microbes that thrive in extremely salty water, as well as microbes that live at temperatures close to boiling. Even more recently, scientists have begun finding archaea in an increasing array of habitats, such as the ocean surface, deep ocean muds, salt marshes, the guts of animals, and even in oil reserves deep below the surface of the Earth. Archaea have gone from obscurity to being nearly ubiquitous in only 25 years! Archaeans have increasingly become the study of scientific investigation. In many ways, archaeal cells resemble the cells of bacteria, but in a number of important respects, they are more like the cells of eukaryotes.

The question arises whether the Archaea are closer relatives of the bacteria or our our group, the eukaryotes. This is a very difficult question to answer, because we are talking about the deepest branches of the tree of life itself; we do not have any early ancestors of life around today for comparison. One novel approach used in addressing the question is to look at sequences of duplicated genes. Some DNA sequences occur in more than one copy within each cell, presumably because an extra copy was made at some point in the past. There are a very few genes known to exist in duplicate copies in all living cells, suggesting that the duplication happened before the separation of the three domains of life. In comparing the two sets of sequences, scientists have found that the Archaea may actually be more closely related to us (and the other eukaryotes) than to the bacteria.

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Archaea: Morphology
enough to distinguish their physical features. You can see archaean images below, made using a variety of micrographic techniques. You might think that organisms so small would not have much variety of shape or form, but in fact archaeal shapes are quite diverse. Some are spherical, a form known as coccus, and these may be perfectly round or lobed and lumpy. Some are rod-shaped, a form known as bacillus, and range from short bar-shaped rods to long slender hair-like forms. Some oddball species have been discovered with a triangular shape, or even a square shape like a postage stamp!

Archaea are tiny, usually less than one micron long (one onethousandth of a millimeter). Even under a high-power light microscope, the largest archaeans look like tiny dots. Fortunately, the electron microscope can magnify even these tiny microbes

Basic Archaeal Shapes : At far left, Methanococcus janaschii, a coccus form with numerous flagella attached to one side. At left center, Methanosarcina barkeri, a lobed coccus form lacking flagella. At

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right center, Methanothermus fervidus, a short bacillus form without flagella. At far right, Methanobacterium thermoautotrophicum, an elongate bacillus form.

Structural diversity among archaeans is not limited to the overall shape of the cell. Archaea may have one or more flagella attached to them, or may lack flagella altogether. The flagella are hair-like appendages used for moving around, and are attached directly into the outer membrane of the cell. When multiple flagella are present, they are usually attached all on one side of the cell. Other appendages include protein networks to which the cells may anchor themselves in large groups. Like bacteria, archaeans have no internal membranes and their DNA exists as a single loop called a plasmid. However, their tRNAs have a number of features that differ from all other living things. The tRNA molecules As with other living things, archaeal cells have an outer cell membrane that serves as a barrier between the cell and its environment. Within the membrane is the cytoplasm, where the living functions of the archeon take place and where the DNA is located. Around the outside of nearly all archaeal cells is a cell wall, a semirigid layer that helps the cell maintain its shape and chemical equilibrium. All three of these regions may be

(short for "transfer RNA") are important in decoding the message of DNA and in building proteins. Certain features of tRNA structure are the same in bacteria, plants, animals, fungi, and all known living things -- except the Archaea. There are even features of archaeal tRNA that are more like eukaryotic critters than bacteria, meaning that Archaea share certain features in common with you and not with bacteria. The same is true of their ribosomes, the giant processing molecules that assemble proteins for the cell. While bacterial ribosomes are sensitive to certain chemical inhibiting agents, archaeal and eukaryotic ribosomes are not sensitive to those agents. This may suggest a close relationship between Archaea and eukaryotes. distinguished in the cells of bacteria and most other living things, but when you take a closer look at each region, you find that the similarities are merely structural, not chemical. In other words, Archaea build the same structures as other organisms, but they build them from different chemical components. For instance, the cell walls of all bacteria contain the chemical peptidoglycan. Archaeal cell 10

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walls do not contain this compound, though some species contain a similar one. Likewise, archaea do not produce walls of cellulose (as do plants) or chitin (as do fungi). The cell wall of archaeans is chemically distinct.
Basic Archaeal Structure : The three primary regions of an archaeal cell are the cytoplasm, cell membrane, and cell wall. Above, these three regions are labelled, with an enlargement at right of the cell membrane structure. Archaeal cell membranes are chemically different from all other living things, including a "backwards" glycerol molecule and isoprene derivatives in place of fatty acids. See text below for details.

The most striking chemical differences between Archaea and other living things lie in their cell membrane. Their are four fundamental differences between the archaeal membrane and those of all other cells: (1) chirality of glycerol, (2) ether linkage, (3) isoprenoid chains, and (4) branching of side chains. These may sound like complex differences, but a little explanation will make the differences understandable. The header for each explanation is color-coded to match the relevant portion of the diagram below. (1) Chirality of glycerol : The basic unit from which cell membranes are built is the phospholipid. This is a molecule of glycerol which has a phosphate added to one end, and two side chains attached at the other end. When the cell membrane is put together, the glycerol and phosphate end of the molecules hang out at the

surface of the membrane, with the long side chains sandwiched in the middle (see illustration above). This layering creates an effective chemical barrier around the cell and helps maintain chemical equilibrium. The glycerol used to make archaeal phospholipids is a stereoisomer of the glycerol used to build bacterial and eukaryotic membranes. Two molecules that are stereoisomers are mirrorimages of each other. Put your hands out in front of you, palms up. Both hands are oriented with fingers pointing away from you, wrists toward you, and with palms upwards. However, your thumbs are pointing different directions because each hand is a mirror image of the other. If you turn one hand so that both thumbs point the same way, that one will no longer be palm-up.

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This is the same situation as the stereoisomers of glycerol. There are two possible forms of the molecule that are mirror images of each other. It is not possible to turn one into the other simply by rotating it around. While bacteria and eukaryotes have D-

glycerol in their membranes, archaeans have L-glycerol in theirs. This is more than a geometric difference. Chemical components of the cell have to be built by enzymes, and the "handedness" (chirality) of the molecule is determined by the shape of those enzymes. A cell that builds one form will not be able to build the other form.

(2) Ether linkage : When side chains are added to the glycerol, most organisms bind them together using an ester linkage (see diagram above). The side chain that is added has two oxygen atoms attched to one end. One of these oxygen atoms is used to form the link with the glycerol, and the other protrudes to the side when the bonding is done. By contrast, archaeal side chains are bound using an ether linkage, which lacks that additional

protruding oxygen atom. This gives the resulting phospholipid different chemical proerties from the membrane lipids of other organisms.

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(3) Isoprenoid chains : The side chains in the phospholipids of bacteria and eukaryotes are fatty acids, chains of usually 16 to 18 carbon atoms. Archaea do not use fatty acids to build their membrane phospholipids. Instead, they have side chains of 20 carbon atoms built from isoprene.

have a different physical structure. Because isoprene is used to build the side chains, there are side branches off the main chain (see diagram above). The fatty acids of bacteria and eukaryotes do note have these side branches (the best they can manage is a slight bend in the middle), and this creates some interesting properties in archaeal membranes. For example, the isoprene side chains can be joined together. This can mean that the two side chains of a single phospholipid can join together, or they can be joined to side chains of another phospholipid on the other side of the membrane. No other group of organisms can form such transmembrane phospholipids. Another interesting property of the side branches is their ability to form carbon rings. This happens when one of the side branches curls around and bonds with another atom down the chain to make a ring of five carbon atoms. Such rings are thought to provide structural stability to the membrane, since they seem to be more common among species that live at high temperatures. They may work in the same way that cholesterol does in eukaryotic cells to stabilize membranes. It's interesting to note that cholesterol is another terpene!

Isoprene is the simplest member of a class of chemicals called terpenes. By definition, a terpene is any molecule bilt by connecting isoprene molecules together, rather like building with Lego blocks. Each isoprene unit has a "head" and a "tail" end (again like a Lego block), but unlike their toy counterparts, isoprene blocks can be joined in many ways. A head can be attached to a tail or to another head end, and tails can be similarly joined. The immense variety of terpene compounds that can be built from simple isoprene units include beta-carotene (a vitamin), natural and synthetic rubbers, plant essential oils (such as spearmint), and steroid hormones (such as estrogen and testosterone). (4) Branching of side chains : Not only are the side chains of achaeal membranes built from different components, but the chains themselves

For more information : An impressive set of links to all things Archaean may be found at Life in Extreme Environments: Archaea on the Astrobiology Web. Or get a general introduction to the major groups of prokaryotes from Kenneth Todar at the University of Wisconsin--Madison. Professor Karl Stetter has created a rich gallery of archaean images on-line at the University of Regensburg's Department of Microbiology.

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More pictures of living archaeans can be seen at the Picture Gallery of the Department of Microbiology, University of Nijmegen, in the Netherlands.

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