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What
I
learned
Some extremophiles have been known for more than
40 years. But the search for them has intensified
recently, as scientists have recognized that places once
assumed to be sterile abound with microbial life. The
hunt has also been fueled in the past several years by
industry's realization that the "survival kits" possessed
by extremophiles can potentially serve in an array of
applications.
Of particular interest are the enzymes (biological
catalysts) that help extremophiles to function in brutal
circumstances. Like synthetic catalysts, enzymes, which
are proteins, speed up chemical reactions without being
altered themselves. Last year the biomedical field and
other industries worldwide spent more than $2.5 billion
on enzymes for applications ranging from the production
of sweeteners and "stonewashed" jeans to the genetic
identification of criminals and the diagnosis of infectious
and genetic diseases. Yet standard enzymes stop
working when exposed to heat or other extremes, and
so manufacturers that rely on them must often take
special steps to protect the proteins during reactions or
storage. By remaining active when other enzymes would
fail, enzymes from extremophiles--dubbed
"extremozymes"--can potentially eliminate the need for
those added steps, thereby increasing efficiency and
reducing costs. They can also form the basis of entirely
new enzyme-based processes. Heat-loving microbes, or thermophiles, are among the
best studied of the extremophiles. Thermophiles
reproduce, or grow, readily in temperatures greater
than 45 degrees Celsius (113 degrees Fahrenheit), and
some of them, referred to as hyperthermophiles, favor
temperatures above 80 degrees C (176 degrees F).
Some hyperthermophiles even thrive in environments
hotter than 100 degrees C (212 degrees F), the boiling
point of water at sea level. In comparison, most
garden-variety bacteria grow fastest in temperatures
between 25 and 40 degrees C (77 and 104 degrees F).
Further, no multicellular animals or plants have been
found to tolerate temperatures above about 50 degrees
C (122 degrees F), and no microbial eukarya yet
discovered can tolerate long-term exposure to
temperatures higher than about 60 degrees C (140
degrees F).
Thermophiles that are content at temperatures up to 60
degrees C have been known for a long time, but true
extremophiles--those able to flourish in greater
heat--were first discovered only about 30 years ago.
Thomas D. Brock, now retired from the University of
Wisconsin-Madison, and his colleagues uncovered the
earliest specimens during a long-term study of microbial
life in hot springs and other waters of Yellowstone
National Park in Wyoming.
The investigators found, to their astonishment, that even
the hottest springs supported life. In the late 1960s they
identified the first extremophile capable of growth at
temperatures greater than 70 degrees C. It was a
bacterium, now called Thermus aquaticus, that would
later make possible the widespread use of a
revolutionary technology--the polymerase chain
reaction (PCR). About this same time, the team found
the first hyperthermophile in an extremely hot and acidic
spring. This organism, the archaean Sulfolobus
acidocaldarius, grows prolifically at temperatures as
high as 85 degrees C. They also showed that microbes
can be present in boiling water.
Brock concluded from the collective studies that
bacteria can function at higher temperatures than
eukarya, and he predicted that microorganisms would
likely be found wherever liquid water existed. Other
work, including research that since the late 1970s has
taken scientists to more hot springs and to environments
around deep-sea hydrothermal vents, has lent strong
support to these ideas. Hydrothermal vents, sometimes
called smokers, are essentially natural undersea rock
chimneys through which erupts superheated,
mineral-rich fluid as hot as 350 degrees C.
To date, more than 50 species of hyperthermophiles
have been isolated, many by Karl O. Stetter and his
colleagues at the University of Regensburg in Germany.
The most heat-resistant of these microbes, Pyrolobus
fumarii, grows in the walls of smokers. It reproduces
best in an environment of about 105 degrees C and can
multiply in temperatures of up to 113 degrees C.
Remarkably, it stops growing at temperatures below 90
degrees C (194 degrees F). It gets too cold! Another
hyperthermophile that lives in deep-sea chimneys, the
methane-producing archaean Methanopyrus, is now
drawing much attention because it lies near the root in
the tree of life; analysis of its genes and activities is
expected to help clarify how the world's earliest cells
survived.
What is the upper temperature limit for life? Do
"super-hyperthermophiles" capable of growth at 200 or
300 degrees C exist? No one knows, although current
understanding suggests the limit will be about 150
degrees C. Above this temperature, probably no
life-forms could prevent dissolution of the chemical
bonds that maintain the integrity of DNA and other
essential molecules.
Not Too Hot to Handle
Researchers interested in how the
structure of a molecule influences
its activity are trying to understand
how molecules in heat-loving
microbes and other extremophiles
remain functional under conditions
that destroy related molecules in
organisms adapted to more
temperate climes. That work is
still under way, although it seems that the structural
differences need not be dramatic. For instance, several
heat-loving extremozymes resemble their heat-intolerant
counterparts in structure but appear to contain more of
the ionic bonds and other internal forces that help to
stabilize all enzymes.
Whatever the reason for their greater activity in extreme
conditions, enzymes derived from thermophilic
microbes have begun to make impressive inroads in
industry. The most spectacular example is Taq
polymerase, which derives from T. aquaticus and is
employed widely in PCR. Invented in the mid-1980s by
Kary B. Mullis, then at Cetus Corporation, PCR is
today the basis for the forensic "DNA fingerprinting"
that received so much attention during the recent O. J.
Simpson trials. It is also used extensively in modern
biological research, in medical diagnosis (such as for
HIV infection) and, increasingly, in screening for genetic
susceptibility to various diseases, including specific
forms of cancer.
In PCR, an enzyme known as a DNA polymerase
copies repeatedly a snippet of DNA, producing an
enormous supply. The process requires the reaction
mixture to be alternately cycled between low and high
temperatures. When Mullis first invented the technique,
the polymerases came from microbes that were not
thermophilic and so stopped working in the hot part of
the procedure. Technicians had to replenish the
enzymes manually after each cycle.
To solve the problem, in the late 1980s scientists at
Cetus plucked T. aquaticus from a clearinghouse
where Brock had deposited samples roughly 20 years
earlier. The investigators then isolated the microbe's
DNA polymerase (Taq polymerase). Its high tolerance
for heat led to the development of totally automated
PCR technology. More recently, some users of PCR
have replaced the Taq polymerase with Pfu
polymerase. This enzyme, isolated from the
hyperthermophile Pyrococcus furiosus ("flaming
fireball"), works best at 100 degrees C.
A different heat-loving extremozyme in commercial use
has increased the efficiency with which compounds
called cyclodextrins are produced from cornstarch.
Cyclodextrins help to stabilize volatile substances (such
as flavorings in foods), to improve the uptake of
medicines by the body, and to reduce bitterness and
mask unpleasant odors in foods and medicines.
Others Like It Cold, Acidic, Alkaline
Cold environments are actually more common than hot
ones. The oceans, which maintain an average
temperature of one to three degrees C (34 to 38
degrees F), make up over half the earth's surface. And
vast land areas of the Arctic and Antarctic are
permanently frozen or are unfrozen for only a few
weeks in summer. Surprisingly, the most frigid places,
like the hottest, support life, this time in the form of
psychrophiles (cold lovers).
James T. Staley and his colleagues at the University of
Washington have shown, for example, that microbial
communities populate Antarctic sea ice--ocean water
that remains frozen for much of the year. These
communities include photosynthetic eukarya, notably
algae and diatoms, as well as a variety of bacteria. One
bacterium obtained by Staley's group, Polaromonas
vacuolata, is a prime representative of a psychrophile:
its optimal temperature for growth is four degrees C,
and it finds temperatures above 12 degrees C too warm
for reproduction. Cold-loving organisms have started to
interest manufacturers who need enzymes that work at
refrigerator temperatures--such as food processors
(whose products often require cold temperatures to
avoid spoilage), makers of fragrances (which evaporate
at high temperatures) and producers of cold-wash
laundry detergents.
Among the other extremophiles now under increasing
scrutiny are those that prefer highly acidic or basic
conditions (acidophiles and alkaliphiles). Most natural
environments on the earth are essentially neutral, having
pH values between five and nine. Acidophiles thrive in
the rare habitats having a pH below five, and
alkaliphiles favor habitats with a pH above nine.
Highly acidic environments can result naturally from
geochemical activities (such as the production of
sulfurous gases in hydrothermal vents and some hot
springs) and from the metabolic activities of certain
acidophiles themselves. Acidophiles are also found in
the debris left over from coal mining. Interestingly,
acid-loving extremophiles cannot tolerate great acidity
inside their cells, where it would destroy such important
molecules as DNA. They survive by keeping the acid
out. But the defensive molecules that provide this
protection, as well as others that come into contact with
the environment, must be able to operate in extreme
acidity. Indeed, extremozymes that are able to work at
a pH below one--more acidic than even vinegar or
stomach fluids--have been isolated from the cell wall
and underlying cell membrane of some acidophiles.
Potential applications of acid-tolerant extremozymes
range from catalysts for the synthesis of compounds in
acidic solution to additives for animal feed, which are
intended to work in the stomachs of animals. The use of
enzymes in feed is already quite popular. The enzymes
that are selected are ones that microbes normally
secrete into the environment to break food into pieces
suitable for ingestion. When added to feed, the enzymes
improve the digestibility of inexpensive grains, thereby
avoiding the need for more expensive food.
Alkaliphiles live in soils laden with carbonate and in
so-called soda lakes, such as those found in Egypt, the
Rift Valley of Africa and the western U.S. Above a pH
of eight or so, certain molecules, notably those made of
RNA, break down. Consequently, alkaliphiles, like
acidophiles, maintain neutrality in their interior, and their
extremozymes are located on or near the cell surface
and in external secretions. Detergent makers in the U.S.
and abroad are particularly excited by alkaliphilic
enzymes. In Japan, where industry has embraced
extremozymes with enthusiasm, much of the research
into alkaliphilic extremozymes has been spearheaded by
Koki Horikoshi of the Japan Marine Science and
Technology Center in Yokosuka.
To work effectively, detergents must be able to cope
with stains from food and other sources of grease--jobs
best accomplished by such enzymes as proteases
(protein degraders) and lipases (grease degraders). Yet
laundry detergents tend to be highly alkaline and thus
destructive to standard proteases and lipases.
Alkaliphilic versions of those enzymes can solve the
problem, and several that can operate efficiently in heat
or cold are now in use or being developed. Alkaliphilic
extremozymes are also poised to replace standard
enzymes wielded to produce the stonewashed look in
denim fabric. As if they were rocks pounding on denim,
certain enzymes soften and fade fabric by degrading
cellulose and releasing dyes.
A Briny Existence
The list of extremophiles does not end there. Another
remarkable group--the halophiles--makes its home in
intensely saline environments, especially natural salt
lakes and solar salt evaporation ponds. The latter are
human-made pools where seawater collects and
evaporates, leaving behind dense concentrations of salt
that can be harvested for such purposes as melting ice.
Some saline environments are also extremely alkaline
because weathering of sodium carbonate and certain
other salts can release ions that produce alkalinity. Not
surprisingly, microbes in those environments are
adapted to both high alkalinity and high salinity.
Halophiles are able to live in salty conditions through a
fascinating adaptation. Because water tends to flow
from areas of high solute concentration to areas of
lower concentration, a cell suspended in a very salty
solution will lose water and become dehydrated unless
its cytoplasm contains a higher concentration of salt (or
some other solute) than its environment. Halophiles
contend with this problem by producing large amounts
of an internal solute or by retaining a solute extracted
from outside. For instance, an archaean known as
Halobacterium salinarum concentrates potassium
chloride in its interior. As might be expected, the
enzymes in its cytoplasm will function only if a high
concentration of potassium chloride is present. But
proteins in H. salinarum cell structures that are in
contact with the environment require a high
concentration of sodium chloride.
The potential applications for salt-tolerant enzymes do
not leap to mind as readily as those for certain other
extremozymes. Nevertheless, at least one intriguing
application is under consideration. Investigators are
exploring incorporating halophilic extremozymes into
procedures used to increase the amount of crude
extracted from oil wells.
To create passages through which trapped oil can flow
into an active well, workers pump a mixture of viscous
guar gum and sand down the well hole. Then they set
off an explosive to fracture surrounding rock and to
force the mixture into the newly formed crevices. The
guar facilitates the sand's dispersion into the cracks, and
the sand props open the crevices. Before the oil can
pass through the crevices, however, the gum must be
eliminated. If an enzyme that degrades guar gum is
added just before the mixture is injected into the
wellhead, the guar retains its viscosity long enough to
carry the sand into the crevices but is then broken
down.
At least, that is what happens in the ideal case. But oil
wells are hot and often salty places, and so ordinary
enzymes often stop working prematurely. An
extremozyme that functioned optimally in high heat and
salt would presumably remain inactive at the relatively
cool, relatively salt-free surface of the well. It would
then become active gradually as it traveled down the
hole, where temperature rises steadily with increasing
depth. The delayed activity would provide more time
for the sand mixture to spread through the oil-bearing
strata, and the tolerance of heat and salt would enable
the enzyme to function longer for breaking down the
guar. Preliminary laboratory tests of this prospect, by
Robert M. Kelly of North Carolina State University,
have been encouraging.
If the only sources of extremozymes were large-scale
cultures of extremophiles, widespread industrial
applications of these proteins would be impractical.
Scientists rarely find large quantities of a single species
of microbe in nature. A desired organism must be
purified, usually by isolating single cells, and then grown
in laboratory culture. For organisms with extreme
lifestyles, isolation and large-scale production can prove
both difficult and expensive.
Harvesting Extremozymes
Fortunately, extremozymes can be produced through
recombinant DNA technology without massive culturing
of the source extremophiles. Genes, which consist of
DNA, specify the composition of the enzymes and
other proteins made by cells; these proteins carry out
most cellular activities. As long as microbial prospectors
can obtain sample genes from extremophiles in nature
or from small laboratory cultures, they can generally
clone those genes and use them to make the
corresponding proteins. That is, by using the
recombinant DNA technologies, they can insert the
genes into ordinary, or "domesticated," microbes, which
will often use the genes to produce unlimited, pure
supplies of the enzymes.
Matt Belanger
cooldude5000@juno.com
3800 Underwood St.
Chevy Chase, MD 20815
United States
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Predators
There are no real predators of extremophiles.
Still extremophiles have to be able to survive.
There are many types of extremophiles.
One is a thermophile.
This can live at the greatest heat of any living thing.
Thermophiles survive by making abnormal changes in their
protien structure.
All extremophiles live at extrem something.
For example extreme heat, coldness, even depth.
Extremophile is not the actual thing.
It is more a group with a bunch of things in it.
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