Download Frontiers in Microbiology

Frontiers in Microbiology is intended to help high school and
college biology teachers update their content knowledge about
this rapidly advancing field. Nine sections describe research
results and current thinking about microbes and their impacts
on the health of humans and the planet.
Table of Contents
1
I. Introduction
3
II. Microbes and the Origin of Life
6
III. Microbes and Extreme Environments
10
IV. Microbes and the Three Domains
13
V. Microbe Communities
15
VI. Microbes’ Influence on Earth
17
VII. Microbial Genomics
20
VIII. Microbes and Humans
24
IX. Microbiology Education
26
References
BSCS Administrative Staff
Rodger W. Bybee, Ph.D., Executive Director
Janet Carlson Powell, Ph.D., Associate Director
Pamela Van Scotter, Director, Center for Curriculum Development
Project Staff
Mark Bloom, Ph.D., Project Director/Writer
Susan Rust, Director of Web Communications
Cody Rust, Graphic Design
Stacey Luce, Production Specialist/Permissions
Publication Services, Editing
BSCS
5415 Mark Dabling Blvd
Colorado Springs, CO 80918
719.531.5550
www.bscs.org
Copyright © 2006 BSCS
Supported by the Department of Energy
Our planet contains creatures that “breathe” metals and can survive extreme temperatures, pH,
salinity, and dehydration. By almost any measure, microbes are vital to the health of the earth.
They had been evolving for nearly 3 billion years before the emergence of the first visible
organisms. It is the microbes that generated the earth’s geochemical cycles and today they
maintain the oxygen in our atmosphere.
Despite their importance, microbes are
second class citizens in biology’s
hierarchy. We tend not to notice microbes
unless they can help us, as in the process of
fermentation, or hurt us, as in the case of
pathogenic species. This explains why
many advances in microbiology are
associated not with basic research but with
industry or healthcare. The rest of the time
microbes are easy to overlook; out of sight,
out of mind. Recent progress in
microbiology however, challenges us to
overcome our bias against the invisible.
The Human Genome Project (HGP) spurred the development of high-throughput DNA
sequencing. In 1995 the first genome of a free-living organism, the bacterium Haemophilus
influenzae, was sequenced. Eleven years later, more than 300 completed microbial genomes are
available and nearly twice that number are in progress. Analyses of these genomes are changing
our concept of the tree of life and expanding our view of where life can be found. A phylogenetic
tree based on molecular data reveals that plants and animals occupy but a couple of twigs on the
branch labeled “Eukarya.”
Techniques such as whole-genome shotgun sequencing enable researchers to investigate
microbial diversity without the need to grow thousands of different species in the laboratory. A
recent study by Craig Venter and coworkers (Venter et al., 2004) sampled the waters of the
Sargasso Sea. Even though this part of the Atlantic Ocean is nutrient poor, Venter’s group found
abundant microbial life. Using whole-genome shotgun sequencing they sequenced DNA from at
least 1,800 different species, including 148 putative new species. They also sequenced more than
1.2 million new genes. Such an approach holds great promise for exploring the largely unknown
boundaries of microbial diversity.
Frontiers in Microbiology
BSCS © 2006
Supported by the Department of Energy
1
High school biology classes convey very
little of the revolution occurring in
microbiology. The vast majority of high
school textbooks treat microbes as they did
a generation ago. Students learn the
anatomy of bacteria; they learn how
bacterial cells differ from the cells of plants
and animals. Little new information about
the origins, ubiquity, diverse metabolisms,
life in communities, and environmental
impacts of microbes is included. At the
same time, textbooks stress the diseases
caused by a small minority of microbial
species. In the context of biotechnology,
bacteria are mentioned as useful for cloning
genes but they are not discussed as subjects
worthy of study in their own right. Students
do not appreciate why governments and
biotechnology companies are investing huge sums of money to understand how microbes
function and affect our health and environment. ■
Frontiers in Microbiology
BSCS © 2006
Supported by the Department of Energy
2
The history of microbes is long indeed. Their appearance on earth predates that of visible
creatures by about 3 billion years. Trying to determine exactly when the first microbes arose is
complicated. Traces of life from so long ago are subtle and difficult to interpret. So called
biosignatures are traces of life left behind in rocks by ancient microbes. They may be
microscopic features found inside the rocks or data derived from isotopic ratios. Unfortunately,
other nonbiological processes can mimic biosignatures. If a particular biosignature is found in an
area that had conditions favorable to life, such as a shallow sea, then that observation strengthens
the case that the biosignature arose from microbes and not through a nonbiological process. This
means that a good understanding of geology is essential to interpret putative ancient microbial
fossils.
One type of biosignature is called a
stromatolite. Stromatolites are layered
dome-shaped formations produced by
ancient bacterial colonies. Some
stromatolites from northwestern Australia
have been dated to 3.5 billion years ago.
Other evidence suggests that microbes arose
even before that time. In 1996 a meteorite
from Mars dated to 3.9 billion years ago was
reported to contain evidence of microbes.
Further analysis suggested that most of this
evidence could be accounted for by other
natural processes. Today, the 3.9 billion year
estimate from the meteorite data has been largely discredited (Simpson, 2003).
Regardless of exactly when the first microbes arose, they evolved out of what is commonly
referred to as the primordial soup. According to this view, as the new earth began to cool,
organic compounds were created through energy supplied by lightening, radiation from the sun,
and the Earth’s own heat. As local concentrations of these organic molecules increased, they
began to polymerize. Eventually, they became autocatalytic and replicated themselves.
Experimental support for this view was provided by Stanley Miller, a graduate student working
in the laboratory of Harold Urey at the University of Chicago in 1953 (Miller, 1953).
Frontiers in Microbiology
BSCS © 2006
Supported by the Department of Energy
3
In Miller’s experiment, he
heated water in a flask,
creating water vapor. This
was his model of the
primordial ocean. The top
of the flask contained
methane, ammonia, and
hydrogen. These gases,
along with the water vapor,
were his atmosphere. He
then subjected the gases to a
continuous electric
discharge (his lightning). As
the gases interacted, they
formed a variety of watersoluble organic compounds,
including amino acids.
This ingenious, yet simple,
experiment demonstrated
that organic molecules
could be spontaneously
created under conditions
thought to be similar to
those of early earth. This
idea was given further
support in 1970 when
extraterrestrial amino acids
were found in a meteorite.
This discovery
demonstrated that chemical
reactions similar to those created by Miller occurred on the meteorite parent body early in the
history of the solar system.
Impacts from colliding asteroids and comets would have added some organic material to the
primordial soup. Nevertheless, the soup would contain just a dilute concentration of organic
molecules. How could this organic broth develop into a living cell? To create life as we know it,
these small organic molecules would have to form larger polymeric molecules and acquire the
ability to replicate themselves. The dilute conditions of the soup would not be
thermodynamically favorable to polymerization. Scientists speculate that clays and metal cations
functioned as chemical catalysts to stimulate these reactions. This scenario would be plausible if
the earth’s surface had cooled and temperatures were low enough to allow weak noncovalent
bonds characteristic of adsorption to form (Bada and Lazcana, 2002). This may have been the
case; scientists believe that 3 billion years ago the sun burned less brightly than it does today.
Frontiers in Microbiology
BSCS © 2006
Supported by the Department of Energy
4
Although DNA is the genetic material in living cells, RNA is a simpler molecule and was more
likely the first type of self-replicating polymer to form. This notion is supported by the discovery
of various types of catalytic RNAs found in cells living today. At the same time that RNA
polymers were forming, other organic molecules were forming colloidal suspensions. Such
suspensions can spontaneously form spherical structures called coacervates. Coacervates are
small, cell-sized bodies with diameters of between 1 and 100 microns. They are bounded by an
arrangement of small organic molecules that resembles the outer membrane of a cell. The first
cells are thought to have arisen after a self-replicating polymer such as RNA became enclosed
within a coacervate.
Within the past decade, some scientists have proposed that the first life on earth was
characterized by a series of self-sustaining chemical reactions based on simple monomeric
compounds derived from carbon monoxide and carbon dioxide. If true, this theory would
describe a form of life unlike that which we know today. Furthermore, such organisms would be
difficult, if not impossible, to study since they left behind no hereditary material to serve as an
historical record. This “metabolist theory” remains mostly speculation. The chemical reactions it
describes have not been shown to be autocatalytic or to take place under prebiotic conditions.
The metabolist and RNA theories of life are not mutually exclusive. If the chemistry described
by the metabolist theory did occur, it could have enriched the prebiotic soup in which RNAbased life was brewing. ■
Frontiers in Microbiology
BSCS © 2006
Supported by the Department of Energy
5
Most of the biodiversity on earth is found among the microbes. They have exploited virtually
every niche where life is possible. Plants and animals by contrast are very finicky, requiring a
relatively narrow range of environmental conditions to survive. Scientists exploring the extreme
environments in which microbes live find that these conditions are not too dissimilar from those
thought to exist elsewhere in the solar system and presumably in many other places throughout
the universe. Indeed, the ability of microbes to thrive under such severe conditions makes the
possibility of microbial life elsewhere in the universe seem rather likely to many scientists.
Some species of microbes can thrive at
high temperatures once thought to be
incompatible with life. For example, hot
springs such as those found in
Yellowstone National Park are teeming
with microbes that live at near-boiling
temperatures. An even more extreme
environment is that found around
hydrothermal vents on the ocean floor.
First discovered in 1977, hydrothermal
vents are underwater geysers. Seawater
is sucked into cracks on the seafloor
where it encounters molten rock, or magma. The hot magma superheats the water, which is
forcibly discharged back into the ocean through a hydrothermal vent. As the mineral-rich
superheated water meets the near-freezing water of the seafloor, minerals precipitate out of
solution and form tall structures called chimneys. A wide variety of organisms such as tube
worms and crabs live near the vents. These animals have no digestive systems but rely on
symbiotic relationships with bacteria to obtain nutrients. Bacteria, rather than photosynthetic
plants, are the producers and initiate the food chain in this ecosystem. Instead of relying on light
energy to power photosynthesis, these thermophilic bacteria oxidize hydrogen sulfide to power
chemosynthesis. Until the discovery of these deep-sea vent ecosystems, it was believed that all
life on earth depended on sunlight.
Microbes that live at high temperatures are not of merely academic interest. Biotechnology
companies often use microbes, or enzymes isolated from microbes, to carry out chemical
reactions needed to produce a product. Since the rate of enzymatic reactions increases with
temperature, microbes that have evolved heat resistance can perform these reactions more
efficiently than microbes adapted to cooler temperatures. The most popular heat-resistant
bacteria used today is Thermus aquaticus. This bacterium thrives at 70ºC (158ºF). It was first
isolated from hot springs in Yellowstone National Park in 1969. Its two discoverers, Thomas
Frontiers in Microbiology
BSCS © 2006
Supported by the Department of Energy
6
Brock and Hudson Freeze from Indiana University, grew cultures of Thermus aquaticus in the
laboratory and sent one to the American Type Culture Collection. This allowed other scientists
from around the world to obtain cultures for their own research. By 1976, other scientists had
isolated a heat- stable DNA polymerase called Taq from Thermus aquaticus.
In the early 1980s, a researcher named Kary Mullis at the Cetus Corporation was working on a
clever way to amplify short DNA sequences. His technique was called the polymerase chain
reaction (PCR). It used a bacterial DNA polymerase to synthesize copies of the DNA to be
amplified. He originally used an enzyme isolated from E. coli. Unfortunately, since his technique
relied on repeated exposure to near-boiling temperatures, the E. coli enzyme was destroyed and
had to be replaced 20 to 30 times during each amplification. This was not only inconvenient but
also very expensive. Mullis realized that using a heat-stable DNA polymerase would eliminate
this problem. After substituting the heat-stable Taq enzyme for the E. coli enzyme, Mullis was
able to add enzyme only at the beginning of the amplification; it would remain suitably active
until the end of the process. Use of the Taq enzyme made PCR simple and affordable. Cetus
rewarded Mullis with a $10,000 bonus and later sold the patent rights for PCR to Roche
Molecular Systems for $300 million. In 1993, Mullis was awarded the Nobel Prize in Chemistry
for his invention of PCR. Today, biotechnology companies spend hundreds of millions of dollars
on Taq polymerase each year.
Just as some microbes are heat-adapted, other
species have adapted to the cold. Scientists
recognize two categories of microbes that live
in cold environments: psychrotolerant and
psychrophilic. Psychrotolerant microbes
prefer living at warmer temperatures but can
survive temporarily under cold conditions.
Psychrophylic microbes actually prefer to live
at cold temperatures, between 0°C and 20°C.
These cold-living microbes consist of various
species of unicellular bacteria, algae, and
fungi. Some of these organisms have been
found in ice samples taken from 3.2
kilometers (two miles) below the earth’s
surface (Carey, 2005). Of course if the
organism freezes, then life processes such as metabolism, growth, and reproduction cannot take
place. Microbes employ a variety of strategies for surviving these harsh conditions. Some
contain specific sugars and proteins that function as an antifreeze, lowering the temperature
needed for ice crystals to form. Other microbes become freeze-dried, forming what appear to be
lifeless spores that retain the ability to spring back to life if they are provided with water and
higher temperature.
Selection pressures to cope with dehydration have led to the appearance of some exotic microbe
species. In 1956, Arthur W. Anderson at the Oregon Agricultural Experiment Station in Corvallis
noticed reddish-colored bacteria growing on some spoiled meat that had been sterilized by
Frontiers in Microbiology
BSCS © 2006
Supported by the Department of Energy
7
exposure to a high dose of radiation. These bacteria, called Deinococcus radiodurans, are the
most radiation-resistant organism known to exist. They can withstand radiation doses 1500 times
the amount that would kill a human. For this reason D. radiodurans has been nicknamed Conan
the Bacterium (Huyghe, 1998). Although scientists are still trying to figure out how these
bacteria survive exposure to extreme radiation, the answer has to do with DNA repair. All living
organisms have DNA repair systems that cope with damage to their DNA caused by radiation
from the sun, chemicals in the environment, and mistakes made by the cell’s replication
machinery. There are limits to how much DNA damage can be repaired. D. radiodurans starts
off with an advantage by having multiple copies of its genes, as compared to most microbes that
have but one copy of each gene. These extra copies serve as backups; when one gene is
damaged, its duplicates can continue to function until the damage is repaired. D. radiodurans
also has more effective DNA repair systems in comparison to other radiation-sensitive microbes.
Why did D. radiodurans evolve this resistance to radiation? In nature, this species has adapted to
surviving long periods without water. Since DNA damage caused by dehydration is similar to
that caused by radiation, D. radiodurans has the ability to cope with both types of environment.
Scientists are finding microbes living in
almost everyplace they look, even in
rocks. Microbes classified as endoliths
live in rocks or in the spaces between
mineral grains. These autotrophs use
iron, potassium, or sulfur as the basis for
their metabolism. In their inhospitable
environment, endoliths can’t afford to
spend much energy on reproduction.
They are thought to undergo cell division
about once every 100 years (Carey,
2005). Endoliths have been found living about three kilometers (two miles) below the earth’s
surface. It is the high temperature deep in the crust, rather than the high pressure, that limits their
ability to survive deep in the earth. How deep they go remains to be seen.
Photosynthetic microbes called hypoliths are found in polar desert environments. They typically
live beneath rocks, where they are protected from ultraviolet radiation and harsh winds. So far,
most hypoliths that have been characterized are associated with quartz, which is translucent and
allows light to reach the microbes.
Microbes have successfully adapted to nearly every environmental niche on earth. This diversity
has generated species that use many different types of metabolism. Increasingly, scientists at
biotechnology companies are harnessing microbes’ metabolisms and putting them to work.
Microbes, sometimes with the introduction of foreign genes, are being used to produce proteins
of industrial or medical usefulness. Other applications include bioremediation and fuel
production. Presently, about 25 percent of the world’s copper production uses bacteria to leach
the ore and help purify the metal. This process takes about two years, but scientists are trying to
speed up the process by using different species of bacteria that can work at higher temperatures.
After the extractable ore has been removed from a mine, attempts are made to reclaim the land
and return it to a more natural state. These efforts are often unsuccessful because the tailings left
Frontiers in Microbiology
BSCS © 2006
Supported by the Department of Energy
8
behind are too acidic to promote plant growth. Bioremediation scientists are using bacteria to
neutralize the soil and help plants reclaim former mining sites.
Our understanding of the range of extreme environments suitable for microbial colonization has
expanded substantially over the past few decades. Today, scientists realize that the conditions of
some of earth’s extreme environments may exist elsewhere in the solar system. Recent insights
into microbial diversity are informing the search for life elsewhere in the universe. Perhaps one
day soon we will have to make room in our taxonomy for extraterrestrial microbes. ■
Frontiers in Microbiology
BSCS © 2006
Supported by the Department of Energy
9
Taxonomy is the theory and practice of
describing, naming, and classifying
earth’s diverse organisms. Some people
regard taxonomy as rigid and
uninteresting. In fact, it is a dynamic
discipline that responds to our everchanging understanding of earth’s
biodiversity. As mentioned previously,
over the past several decades it has
become apparent that the majority of
biodiversity is found among microbes. This realization has caused taxonomists to change their
classification schemes in light of new information.
Carolus Linnaeus is regarded as the father of taxonomy. In 1758, he developed a system whereby
each type of organism was identified by a unique two-word name corresponding to its genus and
species. Linnaeus further grouped organisms into larger categories based on their similarities and
differences. The groups were arranged in a hierarchical fashion. Linnaeus originally used four
classification categories but our modern system uses seven. In the Linnaeus system, species that
share many characteristics are grouped together in the same genus. Likewise, related genera are
grouped together in the same family. This process repeats all the way up to the kingdom
category.
Originally, biologists recognized just two
kingdoms, Plantae (plants) and Animalia
(animals). When microbes were first
identified, they were assigned to one of these
two kingdoms. During the middle of the 19th
century, Ernst Haeckel proposed creating a
third kingdom called Protista. Within the
Protista kingdom were placed unicellular
organisms. Bacteria were a major group
within this kingdom. In the early part of the
20th century, it was recognized that bacteria
differed in fundamental ways from other
organisms with regard to their cell structure.
A fourth kingdom called Monera was created
to set them apart. Finally, in the 1950s,
Robert Whittaker expanded the system still
further by adding a fifth kingdom called
Frontiers in Microbiology
BSCS © 2006
Supported by the Department of Energy
10
Fungi. Viruses are not part of this classification system since they are not composed of cells,
being just nucleic acid surrounded by a protein coat.
Whittaker’s five-kingdom system was widely accepted by biologists but just 20 years later
another sweeping change occurred in taxonomy. Previous classification systems relied on visible
traits to describe and classify organisms. Carl Woese was analyzing invisible traits, namely DNA
sequences. Specifically, he compared sequences from a gene for ribosomal RNA across many
species. Woese realized that an organism’s DNA provided an historical record of its evolution.
By comparing sequences across species he could describe evolutionary relationships independent
of the traditional use of visible traits. Evolutionary trees constructed from molecular data
generally yield identical or similar trees to those constructed from morphological data. This
agreement represents some of the best proof supporting the theory of evolution.
Nevertheless, comparisons of the rDNA sequences did produce some surprises. Woese found
that most of the sequence diversity occurred within the microbes. Analysis of this data suggested
to Woese that a new category of classification, the domain, was needed (Woese et al., 1990). The
rDNA sequences from bacteria fell into two distinct groups—the true bacteria and the ancient
bacteria (meaning those thought to be living fossils of the first bacteria to have evolved on earth).
These two groups of microbes are as different from one another as either are to eukaryotic
organisms. Woese proposed a classification system that featured three domains called Bacteria,
Archaea, and Eukarya. Domain Bacteria includes the true bacteria. Domain Archaea includes all
of the ancient bacteria. For example, methanogenic bacteria are members of the Euryarchaeota
branch of the Archaea. They are strictly anaerobic and tend to be heat tolerant, suggesting an
ancient origin. Today, there are confined to environments such as cow intestines and the soils
beneath flooded rice paddies. Domain Eukarya includes all the organisms within the four
eukaryotic kingdoms—Animalia, Plantae, Protista, and Fungi. An evolutionary tree based on
Woese’s three-domain classification system shows the dominance of microbes on earth. Plants
and animals are depicted as a couple of twigs on the tree.
Molecular sequence data are increasingly being used to investigate evolutionary relationships
(Driskell et al. 2004). After two species diverge from each other, they begin to collect sequence
mutations independent of one another. This means that two species that are closely related to
each other will have DNA (or amino acid) sequences that are more similar to each other than two
species that are more distantly related. Such sequence analysis can be used to construct family
trees of organisms. This data is not without its drawbacks however.
Frontiers in Microbiology
BSCS © 2006
Supported by the Department of Energy
11
The assumption made in
phylogenetics is that DNA
sequences are passed on
only from parent to
offspring. This is usually
an accurate assumption.
Microbes however present
a more complex situation.
Many species of microbes
can pass on genetic
information not just to
their progeny, but also to
other individuals in the
same population or even
to members of other
species. This is called
horizontal transfer or
lateral transfer. When
horizontal transfer occurs,
the introduced gene brings
into the cell its own
history, which does not
reflect that of the host cell.
An extreme case of
horizontal transfer took
place about one billion
years ago when Eubacteria
entered into a symbiosis
with Archaea-type host
cells that led to the
development of some
organelles (mitochondria
and chloroplasts). It
remains to be seen just how much horizontal transfer will limit our ability to tease out
evolutionary relationships among the microbes. ■
Frontiers in Microbiology
BSCS © 2006
Supported by the Department of Energy
12
When we think of microbes we typically envision them as free-living cells swimming in water or
resting in the soil. Scientists have learned most of what they know about microbes by isolating
them and growing cultures of homogeneous populations in the laboratory. This approach has
taught us a great deal but also leaves out some of the more interesting and important aspects of
microbial life. In nature, microbes don’t often lead a solitary existence. Instead their populations
often reside in or on other organisms. They may share an environment with other species of
microbes with which they compete. It has been estimated that approximately 99 percent of
bacteria live in microbial communities and not in a free-living state. This fact alone illustrates
why it is critical to study microbial communities.
Bacteria within a population can
communicate with each other by sending
and receiving chemical signals. One
important type of microbe community is
called a biofilm. Unless the population of
bacteria is sufficiently large, forming a
biofilm doesn’t benefit the individual cells.
Bacteria can detect the presence of their
neighbors through a process called quorum
sensing. This process works through
secreted molecules called autoinducers.
When the concentration of autoinducers reaches a threshold, the formation of a biofilm begins.
To form a biofilm, the bacteria first attach to a surface that has nutrients. They next secrete a glue
that secures their attachment. During this time gene expression is changing. Since the need for
movement is no longer important, genes associated with the flagellum are turned off and genes
associated with pili are turned on. The bacteria begin to pile up on each other. Clusters of cells
become separated from each other by channels of water. This arrangement is in some ways
similar to a rudimentary circulatory system. Nutrients make their way to cells of the biofilm
through these channels while waste products are removed via the same channels. Some cells in
the biofilm are not located near these channels and rely instead on diffusion to obtain nutrients
and expel waste. Cells buried deep within the biofilm may be deprived of oxygen and remain
dormant. Under these conditions, the bacteria comprising the biofilm express different subsets of
their genome depending on their local environment. In this way, a biofilm can be compared to a
multicellular organism.
Understanding biofilms is important for recognizing how bacteria interact with our bodies and
how to treat infections by pathogenic bacteria. Some biofilms are relatively innocuous. They
Frontiers in Microbiology
BSCS © 2006
Supported by the Department of Energy
13
form coatings on our teeth and even on contact
lenses. Other biofilms are life-threatening. Patients
with the disease cystic fibrosis cannot properly
regulate the passage of salt in and out of their cells.
A mucus forms in the lung tissue, which provides a
breeding ground for bacteria. Some species such as
Pseudomonas aeruginosa can form biofilms in lung
tissue.
It is much more difficult to rid the body of bacteria
organized into a biofilm as compared to those in the
free-living state. First, the sheer size of the biofilm
is too large for the macrophages of the immune
system to cope with. Second, treatment with
antibiotics can be problematic. It is more difficult
for antibiotics to reach and destroy cells deep
within the biofilm. The drug may kill cells on the
outer surface, leaving the dormant cells of the
interior to reactivate the infection once the
antibiotics are gone. Bacteria such as Escherichia
coli and Pseudomonas aeruginosa have been
shown to form biofilms upon exposure to low
levels of antibiotics (Hoffman et al., 2005).
Presumably this response evolved as a result of microbial competition.
Biofilms can be helpful or harmful. Biofilms have been harnessed to treat sewage and
decontaminate ground water. They have also been used to produce biochemicals used in
medicines, cleaning products, and food additives. On the other hand, biofilms cost water-based
industries billions of dollars per year in lost productivity and damage to product and capital
infrastructure. Industries that are seriously impacted by biofilms include
•
•
•
•
•
•
•
medicine (devices and implants);
food processing;
paper manufacturing;
oil recovery;
drinking water;
cooling water; and
shipping (from biofilms forming on the hulls of ships).
The reductionism that has characterized microbiology for decades has greatly enhanced our
understanding of microbes. As discussed in this section, however, there are limits to how far this
approach can take us toward understanding microbial communities. The genomes of many
microbes have been completely sequenced. We are now in a position to study microbiology
using a more holistic perspective. The complexity of microbial communities and their
relationships to other organisms and the environment requires scientists to adopt a
multidisciplinary systems-biology approach. ■
Frontiers in Microbiology
BSCS © 2006
Supported by the Department of Energy
14
The impacts of microbes on earth’s
environment have been and continue
to be substantial. The sheer number
of microbes on Earth is staggering. It
has been estimated that we share the
planet with some 5 × 1031 microbes,
which weigh more than 50
quadrillion metric tons. Put another
way, microbes constitute about 90
percent of the earth’s biomass,
excluding cellulose, and more than
60 percent of the biomass when
cellulose is included (ASM, 2004).
These diverse microbes are responsible for cycling the elements essential for life, such as
oxygen, nitrogen, carbon, sulfur, and hydrogen. By making these elements available in soils,
microbes increase its fertility and allow for plant growth. The plants, in turn, are the producers of
our ecosystem, providing the energy needed by animals, including humans. Microbes also cycle
the gases in our atmosphere. We tend to think of green plants as being responsible for providing
us with oxygen through photosynthesis. Actually, more photosynthesis is carried out by microbes
than by plants. Ultimately, all photosynthesis derives from microbes. The cyanobacteria
(formerly called blue-green algae) can live aerobically or anaerobically. They are responsible for
the initial rise of atmospheric oxygen around 2.3 billion years ago (Kastling & Siefert, 2002).
Eukaryotoic algae and land plants acquired their photosynthetic capabilities from cyanobacteria
through endosymbiosis. This conclusion is based on sequence analysis of ribosomal RNA and
chloroplast DNA.
Microbes are also major recyclers of other atmospheric gases that contribute to the earth’s
“greenhouse effect.” Theses gases, including water vapor, carbon dioxide, nitrous oxide,
methane, and ozone, allow light energy from the sun to reach the earth. This energy is absorbed
by the earth’s surface and re-radiated as heat. The greenhouse gases then absorb much of this
heat and re-radiate it in all directions. This means that less heat escapes into outer space and the
earth stays warmer than it would otherwise. If the greenhouse gases were not present, the
average temperature of the earth would fall by some 60°F. However, it is possible to have too
much of a good thing. Today, excessive quantities of greenhouse gases are increasing the
average temperature of the earth. This problem is called global warming and threatens the
delicate balance of life on this planet.
Frontiers in Microbiology
BSCS © 2006
Supported by the Department of Energy
15
Ecosystems are so complex that researchers often resort to using modeling techniques that take
into account the many interrelationships found within the ecosystem. Sometimes models produce
unexpected results. For example, a recent study (Knight, 2004) found that the production of acid
rain can actually work to lessen global warming. A significant portion of the human-induced
global warming comes from agriculture. For example, microbes that use hydrogen and acetate
found in peat produce methane. Living alongside the methane-producing microbes are others that
metabolize sulfur. The sulfur-eating microbes can outcompete the methane-producing ones,
thereby reducing the amount of methane released into the atmosphere. This study demonstrates
the importance of accounting for sulfur pollution when estimating methane emissions. It does
not, however, suggest a strategy for controlling methane emissions. After all, acid rain upsets the
chemical balance of rivers and lakes, killing fish and damaging trees.
Nitrogen is an element essential to support the
lives of all life. It is a required component of
proteins, nucleic acids and other cellular
components. Although the atmosphere is nearly
80 percent nitrogen, this gas is unavailable for
use by most organisms. The strong bonds
between the two nitrogen atoms render the N2
molecule almost inert. In order for the nitrogen to
be useful for growth, it must first be combined or
“fixed” in the form of ammonium or nitrate ions.
Some species of bacteria can convert nitrogen
gas to ammonia through the process of nitrogen
fixation. Other species can transform ammonia to
nitrate. Still other species decompose organic
matter, releasing fixed nitrogen in the process. Of
special interest are the species of nitrogen-fixing bacteria that form symbiotic relationships with
plants of the legume family. Legumes such as peas and beans have root nodules that contain
Rhizobium bacteria. These microbes supply the plants with fixed nitrogen, reducing the need for
farmers to use commercial fertilizers. ■
Frontiers in Microbiology
BSCS © 2006
Supported by the Department of Energy
16
Microbiologists have known for a long time that the small number of microbes that had been
cultured in the laboratory represents but a tiny fraction of the existing microbial diversity. Over
the past 10 years, new types of laboratory and computer analyses have given researchers tools to
more accurately estimate the numbers and types of microbes in our surroundings. GenBank (the
sequence database hosted by the National Center for Biotechnology Information) now includes
data from over 30,000 different bacterial species. Recent estimates suggest that the sea may
contain more than 2 million bacterial species and a ton of soil may contain 4 million species
(NCBI, 2004). Similar species diversity is likely among the Archaea.
Traditionally, scientists have studied
microbes by obtaining samples from the
environment and taking them back to the
lab, where, by trial and error, they attempt
to identify growth conditions that allow the
microbes to be cultured indefinitely. Not
only is this procedure time-consuming and
costly, but it also excludes many species of
microbes for which suitable growth
conditions cannot be found. New
techniques associated with genomics are giving scientists approaches to studying microbes in
their natural environments.
Often, scientists investigating microbial diversity focus on the
genes encoding the 16S ribosomal RNA. This gene was
selected because it has been conserved across vast taxonomic
distances, yet still shows some sequence variation among
closely related species. After data are obtained from
environmental samples, the DNA sequences are compared to
each other and to all known 16S rRNA genes. Such studies
have found new uncultured species even in well-studied
environments such as the human mouth.
One scientist, J. Craig Venter, began an ambitious two year
project in August 2003 that is attempted to assess the microbial
diversity of the world’s oceans (Shreeve, 2004). Aboard his
yacht, the Sorcerer II, Venter sailed a route similar to that taken
by Charles Darwin aboard the HMS Beagle in the 19th century.
After every 200 miles, the crew pumped 53 gallons of seawater
taken from a depth of about five feet on board the yacht. The
Frontiers in Microbiology
BSCS © 2006
Supported by the Department of Energy
17
seawater was forced through a series of filters to collect the microbes. The filters were then sent
back to Venter’s lab in Rockville, Maryland for analysis.
Once in the lab, the filters were treated to remove everything but microbe DNA. This DNA,
which represented all the different microbe species that were collected on the filter was then
forced through a pinhole under pressure to generate a series of millions of shorter DNA
fragments. The fragments were amplified using PCR and subjected to DNA sequencing
reactions. The resulting DNA sequences were analyzed by computer. The computer algorithms
searched for overlapping sequences to create longer ones. Using bioinformatics tools, the number
of species was estimated by comparing the sample DNA to that from known microbial species.
Venter chose the Sargasso Sea near Bermuda as the site for a pilot test of his approach. This area
of the ocean was chosen because it is low in nutrients and thought to be somewhat of an oceanic
desert. To his surprise, everywhere he went the samples were teeming with microbial life. In a
report of his Sargasso Sea results, Venter (2004) reported that his group sequenced more than 1
billion bases. The DNA sequences represented approximately 1800 species, including 148
unknown groups. About 1.2 million new genes were found. A follow-up study conducted in
Long Island Sound identified DNA sequences from nearly 1000 different species. Only 1 percent
of the Long Island species overlapped with those from the Sargasso Sea (Pennisi, 2004).
Although Venter’s study concentrates on bacteria, his approach should also work for
investigating the prevalence of plasmids, phages, viruses, and eukaryotic microbes.
An even more recent analysis of soil
microbes has suggested that the
inferred microbial diversity in these
environments has been greatly
underestimated. It has long been known
that if the DNA from a single organism
is heated, the double helix melts and
the two strands separate. If the sample
is allowed to slowly cool, then the
DNA strands will reassociate or
reanneal. Larger and more complex
genomes require longer times for this reannealing to occur than do smaller genomes. Such an
approach has been used for decades to estimate the size and complexity of genomes from diverse
organisms. About 15 years ago, Torsvik and coworkers recognized that pooled genomic DNA
from a microbial community could reanneal like that from a single large genome. Indeed, when
they isolated DNA from a soil microbe community, it reannealed slowly, much like DNA from
an organism with a genome about 7000 times that of a typical bacterium (Torsvik, Goksoyr, &
Daae, 1990). This suggested that the sample may have contained the genomes of 7000 different
taxa.
In 2005, Gans and coworkers realized that the pattern of DNA reassociation reflects the
underlying diversity of the microbial community. They applied new mathematical analyses to
existing data from bacterial communities and came to a startling conclusion. Each 10 grams of
Frontiers in Microbiology
BSCS © 2006
Supported by the Department of Energy
18
healthy soil contains 10 million different bacterial species. Most of this diversity is found among
rare bacteria that are present in small numbers (Gans, Wolinsky, & Dunbar, 2005).
The overwhelming number of species found within microbial communities makes it impossible
to study them using bacteria cultured in the laboratory. It is estimated that 99 percent of the
bacterial species found in the soil cannot be cultured in the laboratory (Gewin, 2006).
Metagenomics is the name given to the study of gene function and interaction regardless of
species within large microbial communities. Researchers today rely on methods of genomics to
study the diversity and interactions that take place within these communities. The use of wholegenome shotgun sequencing gives an indication of the diversity that resides within a microbial
community. It also allows scientists to study the community in its entirety, almost as though it
were a single organism. ■
Frontiers in Microbiology
BSCS © 2006
Supported by the Department of Energy
19
If we consider a human being to be a
collection of cells, then we are all 90
percent microbial! This conclusion is
based on the estimate that within our
bodies there are about 10 microbes for
each human cell (ASM, 2004).
Connections between human health and
microbiology go all the way back to the
discovery of microbes. The amateur Dutch scientist Antonie van Leeuwenhoek is credited with
being the first to observe microbes in the late 17th century (DeKruif, 1926). Leeuwenhoek first
observed microbes swimming in rainwater. He wondered if these “wretched beasties” fell from
the sky or had a more earthly origin. To find out he performed a simple experiment. He
scrupulously cleaned a porcelain bowl and set it out in the rain. He was careful to place the bowl
up off the ground so that the splashing raindrops would not introduce dirt into the bowl. When he
examined the fresh rainwater under his microscope, there were no beasties to be seen. He let the
rainwater sit for several days and when he looked again, there they were. Thus, Leeuwenhoek
demonstrated that the microbes did not come from the rain but from the earth.
Leeuwenhoek was also interested in how microbes interacted with humans. In one of his first
investigations he examined some material taken from between
his teeth and was fascinated to see more of his little beasties.
Later, he encountered an old man with especially bad teeth.
The old man explained to Leeuwenhoek that he had never in
his life cleaned his teeth. Delighted to hear this, Leeuwenhoek
persuaded the old man to come to his study so he could obtain
samples to view through his microscope. He witnessed quite a
show. Today, scientists estimate that the human mouth contains
more than 700 different species of microbes (Pennisi, 2005).
More than half of these species cannot be cultured in the
laboratory.
Even humans who practice good hygiene are full of microbes.
We don’t start out that way however. During the nine months
we spend in our mother’s womb, we exist in a sterile
environment. Not until we are born do we first encounter
microbes: from our mothers, hospital personnel, and our
surroundings. Then, very quickly, microbes begin to colonize
our bodies in very specific ways. Many of these microbes
perform activities that enhance the health of the individual. For
Frontiers in Microbiology
BSCS © 2006
Supported by the Department of Energy
20
example, microbes help digestion, synthesize nutrients, detoxify ingested toxins, kill invading
bacteria, and help organs to grow larger. Animals that are raised in sterile environments to
prevent exposure to microbes are less healthy than their microbe-colonized counterparts.
Some areas of human anatomy
are home to enormous numbers
of microbes, while others
remain sterile. As shown in
Table 1, the human body hosts
approximately 1.25 kilograms
(2.75 pounds) of microbes.
This corresponds to more than
1 × 1014 individual cells.
Aspects of these various
microbe communities are
described in Table 2.
Although microbes are important to our health, a small number of species can cause disease.
Bubonic plague, cholera, syphilis, tuberculosis, dysentery, typhoid fever, and diphtheria are
among the bacterial diseases that affect humans. How do we know when a microbe is the cause
of a disease? Since the late 19th century, medical researchers have used criteria developed by the
German physician Robert Koch (see Table 3) to establish that a particular microbe causes a
specific disease. More recent findings in microbiology are suggesting that Koch’s gold standard
may sometimes be difficult or impossible to achieve.
Frontiers in Microbiology
BSCS © 2006
Supported by the Department of Energy
21
Take an upset stomach for example. We often attribute it to food poisoning. We know that a
number of common species of bacteria such as Salmonella and E. coli can cause food poisoning.
Stomachaches associated with ulcers, however, were long considered to be different. Until fairly
recently, the prevailing wisdom was that stomach ulcers resulted from too much acid in the
stomach, which was brought on by stress. The accepted treatment consisted of medication to
neutralize stomach acid and the introduction of a bland diet.
This stress-related view
of stomach ulcers was
challenged by two
Australian scientists in
1982 (Blaser, 1996).
Since Robert Koch’s
time, scientists have
seen bacteria associated
with stomach ulcers.
The bacteria were not
thought to cause the
ulcers, however. It was believed that the acidic conditions of the stomach would not support the
growth of bacteria. Those bacteria that were observed were thought to result from contamination
of the stomach samples.
An Australian pathologist named J. Robin Warren had examined many gastric biopsies. He
noticed that bacteria were always present in inflamed stomach tissues. Furthermore, the number
of bacteria seen was correlated with the extent of the inflammation. He discussed his
observations with a colleague named Barry Marshall. The two set out to isolate the bacteria and
satisfy Koch’s postulates.
They tried to culture bacteria taken from stomach biopsies for more than one year without
success. Then a fortunate accident occurred. It was their practice to incubate the bacterial
cultures for two days and then look for growth. This time the Easter holiday interfered with their
routine and the cultures were incubated for six days. When they finally removed the culture
plates from the incubator and examined them, they saw vigorous bacterial growth. The bacteria
were Helicobacter pylori, which require
longer growth times than bacteria
typically cultured in the laboratory.
The two researchers were delighted
with their serendipitous discovery. It
did not, however, establish H. pylori as
the cause of stomach ulcers. Koch’s
postulates were not satisfied. In a bold
and perhaps ethically questionable
move, Marshall and another volunteer
Frontiers in Microbiology
BSCS © 2006
Supported by the Department of Energy
22
intentionally ingested cultures of H. pylori. Both developed stomach inflammation and H. pylori
were successfully isolated from their stomach biopsies. A firm link was established between the
bacteria and gastritis. Since neither scientist developed a stomach ulcer, that link remained
unproven. Eventually, epidemiological studies confirmed that people with H. pylori infections
were more likely to have stomach ulcers. The link was further strengthened when antibiotic
treatments cured the ulcers. Eventually, in 2005, the two researchers received the Nobel Prize in
medicine.
This example illustrates why Koch’s postulates may not be the best criteria for establishing a
microbe as the cause of a disease. We cannot culture many microbes (and potential pathogens) in
the laboratory. In the case of H. pylori, the two scientists were lucky to discover that the bacteria
were slow-growing. In other cases, pathogenic microbes may live as part of a complex
community that doesn’t permit them to be grown as pure cultures in the lab. As discussed earlier,
newer techniques such as PCR have given scientists ways to establish links between a disease
and a specific microbe, even when it can’t be grown in the lab.
Until the advent of antibiotics in the 1940s, bacterial infections were major killers, even in
developed countries. Antibiotics were hailed as magic bullets, able to specifically obliterate
disease-causing microbes. The first commercially developed antibiotic was penicillin. Within a
few years of its introduction, bacteria resistant to the drug were observed. In fact bacteria
resistant to antibiotics have been around long before their use in medicine. Scientists have found
antibiotic-resistant bacteria in samples taken from artic glaciers. These bacteria were estimated to
be more than 2000 years old.
Bacteria resistant to an antibiotic can pass on copies of their resistance gene to other bacteria
through horizontal transfer. Over the past 50 years, antibiotics have been so widely used that
infections once treatable by antibiotics are killing patients even in the United States. According
to a Centers for Disease Control and Prevention (CDC) estimate, each year nearly two million
people acquire a bacterial infection while in a hospital, resulting in approximately 90,000 deaths
(Bren, 2003). Most of these bacteria are resistant to at least one of the antibiotics normally used
to treat them.
The overuse of antibiotics contributes to the proliferation of resistant bacteria. Common
infections such as colds and flus are caused by viruses. Antibiotics have no effect on them.
Nevertheless, when people with colds or flus visit their doctor, they often receive prescriptions
for antibiotics. To time-pressed physicians it is sometimes easier to just write the prescription
that the patient wants rather than to explain why the antibiotic will not help. The CDC estimates
that one-third of the outpatient prescriptions for antibiotics written in the United States are
unnecessary (CNN.com Health, 2000). The problem of antibiotic-resistant bacteria must be
addressed through education and research. The public needs to realize that the inappropriate use
of antibiotics is dangerous. When prescribed for viral infections, antibiotics are of no benefit and
contribute to the proliferation of resistant bacteria. For similar reasons, farmers need to restrict
their use of antibiotics in animal feed. Finally, researchers need to carry on their quest to develop
more and better antibiotics. ■
Frontiers in Microbiology
BSCS © 2006
Supported by the Department of Energy
23
As discussed in the previous sections, microbiology is in the midst of an exciting revolution.
Microbes have been in the public eye over the past decade like never before. Unfortunately, this
increased awareness has often been passed through a prism of negativity. Fears of bioterrorism
and the emergence of diseases such as AIDS, West Nile, and SARS have reinforced the negative
aspects of microbes. Large-scale incidents of food contamination in developed countries have
reinforced this fear of microbes. An increasing array of products with antimicrobial properties is
available to make us safer, or at least feel safer.
If the current revolution in microbiology
is to reach its full potential, then
microbiology education must fulfill its
promise. Education is important, not
only to train the next generation of
microbiologists, but also to generate an
informed public that can make rational
decisions regarding issues that involve
microbes. Decision makers at all levels
need to be aware of how microbes affect
society. They need to help schools
update and improve their curricula to better teach these important concepts.
Microbes occupy a central position in the tree of life. They can be used as a central organizing
principle in the study of biology. This argues for integrating microbiology into all aspects of
biology education, rather than relegating it to a separate course. Most of the biology content
found in the National Science Education Standards can be taught using microbial examples.
Microbiology provides interesting case studies (historical as well as contemporary) that are
engaging to students and reflect important nature-of-science concepts.
A recent report issued by the National
Research Council (Singer, Hilton, &
Schweingruber, 2005) has reaffirmed the
importance of laboratory-based instruction
while at the same time criticizing the current
state of lab teaching in America’s high
schools. The report found that most lab
activities were included almost as
afterthoughts. They were not integrated into
the rest of the science class. Furthermore,
many high school lab activities do not reflect
Frontiers in Microbiology
BSCS © 2006
Supported by the Department of Energy
24
the process of scientific inquiry. They don’t help students understand how scientists use lab
investigations to explore new areas. Although there was much to criticize regarding the design
and implementation of high school laboratories, the report also acknowledged that teachers face
a number of constraints that limit their ability to provide proper lab experiences to their students.
These can be summed up as time, safety, and money.
Microbiology offers potential solutions to the problems associated with lab-based teaching.
Harmless microbes are easily obtainable and affordable. They are small and reproduce quickly,
which means that they can be used to address concepts of genetics and population biology that
are otherwise difficult to approach experimentally. Although microbiology makes use of cuttingedge technology, its basic tools are simple, inexpensive, and remain relevant to topics of current
interest in biology. Furthermore, many microbiological techniques can be performed within the
brief periods of time allotted for science classes.
Microbiology is well suited to an interdisciplinary
approach. Microbial diversity is best understood by
investigating the geology and chemistry of earth’s
many ecosystems. Mathematics is important for
understanding population growth and changes in
frequency brought about through natural selection,
and for modeling the impacts of microbes on the
environment. Microbiology also provides engaging
case studies that can be incorporated into health,
history, and social science classes. It is also worth
noting that microbiology provides wide-ranging
career opportunities, not just for students interested
in biology but for those with interests in computer
science, mathematics, physical sciences, and
engineering as well.
Thirty years ago new techniques for manipulating DNA led to the explosive growth of the field
of molecular biology. Today, there is virtually no area of biology that has not been greatly
influenced by molecular genetics. Microbiology was well suited to benefit from this scientific
revolution. The small sizes of microbial genomes mean that genomic techniques have (and
continue to) affect microbiology faster and more deeply than most other areas of biology.
Hopefully, decision makers from industry, government, and academia will recognize the
importance of revitalizing microbiology education and will make resources available to meet this
important challenge. ■
Frontiers in Microbiology
BSCS © 2006
Supported by the Department of Energy
25
American Society of Microbiology. (2004) Microbiology in the 21st century: Where are we and
where are we going? (2004) Washington, DC: Author.
Bada, J.L. & Lazcana, A. (2002) Some like it hot, but not the first biomolecules, Science, 296,
1982–1983.
Blaser, M.J., (1996, February). The bacteria behind ulcers, Scientific American, 274(2), 104–107.
Bren, L., (2003, September). Battle of the bugs: Fighting antibiotic resistance. FDA Consumer
Magazine, 36(4). Retrieved January 25, 2006, from
http://www.fda.gov/fdac/features/2002/402_bugs.html.
Carey, B., (2005, February 7). Wild things: The most extreme creatures, LiveScience, Retrieved
Jnauary 25, 2006, from http://www.livescience.com/animalworld/050207_extremeophiles.html
CNN.com Health, (2000, June 1). CDC expands campaign against overuse of antibiotics,
Retrieved from, http://archives.cnn.com/2000/HEALTH/06/01/antibiotic.overuse/
DeKruif, P. (1926). Microbe hunters. Orlando, FL: Harcourt Brace & Company.
Driskell, A.C., Ané, C., Burleigh, J.G., McMahon, M.M., O’Meara, B.C., & Sanderson, M.J.
(2004, November 12). Prospects for building the tree of life from large sequence databases.
Science, 306, 1172–1174.
Gans, J., Wolinsky, M. & Dunbar, J. (2005, August 26). Computational improvements reveal
great bacterial diversity and high metal toxicity in soil. Science, 309, 1387–1394.
Gewin, V. (2006, January 26). Discovery in the dirt. Nature, 439, 384–386.
Hoffman, L.R., D’Argenio, D.A., MacCoss, M.J., Zhang, Z., Jones, R.A., & Miller, S.I. (2005,
August 25). Aminoglycoside antibiotics induce bacterial biofilm formation. Nature, 436, 1171–
1175.
Huyghe, P. (1998, July/August). Conan the bacterium. The Sciences, 38(4), 16–19.
Kasting, J.F. and Siefert, J.L. (2002, May 10). Life and the evolution of earth’s atmosphere.
Science, 296, 1066–1068.
Frontiers in Microbiology
BSCS © 2006
Supported by the Department of Energy
26
Knight, W. (2004, August 3). Acid rain limits global warming. NewScientist.com News Service,
Retrieved January 25, 2006, from http://www.newscientist.com/article.ns?id=dn6231
Miller S. L. (1953). Production of amino acids under possible primitive earth conditions.
Science, 117, 528.
National Center for Biotechnology Information. (2004, March). Microbial diversity: Let’s tell it
how it is. Coffee Break, chapter 622. Retrieved January 25, 2006 from
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?call=bv.View..ShowSection&rid=coffeebrk.chapter.
622
Pennisi, E. (2005, March 25). A mouthful of microbes. Science, 307, 1899–1901.
Pennisi, E. (2004, June 11). Surveys reveal vast numbers of genes. Science, 304, 1591.
Shreeve, J. (2004, August). Craig Venter’s epic voyage to refine the origin of species, Wired,
12(8). Retrieved January 25, 2006, from http://www.wired.com/wired/archive/12.08/venter.html
Simpson, S. (2003, April). Questioning the oldest signs of life. Scientific American, 288(4), 70–
77.
Singer, S. Hilton, M.L., & Schweingruber, H..A. (Eds.) (2005). America’s lab report:
investigations in high school science. Washington, DC: National Academies Press.
Torsvik, V., Goksoyr, J., & Daae, F.L. (1990, March). High diversity in DNA of soil bacteria.
Applied and Environmental Microbiology. 56(3), 782–787.
Venter, C. J., Remington, K., Heidelberg, J.F., Halpern, A.L., Rusch, D., Eisen, J.A., Wu, D.,
Paulsen, I., Nelson, K.E., Nelson, W., Fouts, D.E., Levy, S., Knap, A.H., Lomas, M.W., Nealson,
K., White, O., Peterson, J., Hoffman, J., Parsons, R., Baden-Tillson, H., Pfannkoch, C., Rogers,
Y.H., & Smith, H.O. (2004, April 2). Environmental genome shotgun sequencing of the Sargasso
Sea. Science, 304, 66–74.
Wilson, M. (2005). Microbial inhabitants of humans: Their ecology and role in health and
disease. New York: Cambridge University Press.
Woese, C.R., Kandler, O., & Wheelis, M.L. (1990). Towards a natural system of organisms:
Proposal for the domains Archaea, Bacteria, and Eucarya. Proceedings of the National Academy
of Sciences, 87, 4576–4579.
■
Frontiers in Microbiology
BSCS © 2006
Supported by the Department of Energy
27