Chapter 1 - The Relevance and History of

Microbiology
1 - 1 Introduction

This book will focus on the biology of small things, but what is microbiology?
Microbiology could be defined as the study of organisms too small to be seen with the
naked eye. Figure 1-14 [1] shows the relative size of microbes compared to other living
things. However, the recent discovery of bacteria [2] of near 1 µm in size has made this
definition somewhat inaccurate and in the grand tradition of science, a new definition is
in order.

Figure 1-14 The relative size of microbes

Though microbes are small, they nevertheless span a large range of sizes from the
smallest bacterial cells at ~0.15 µm to giant bacteria larger than 700 µm. The viruses
depicted at the far left of the scale are even smaller.

We will consider microbiology to be the study of organisms that can exist as single
cells, contain a nucleic acid genome for at least some part of their life cycle, and are
capable of replicating that genome. This broad description encompasses an
understandably large group of organisms including fungi [3], algae, protozoa [4] and
bacteria. Examples of these are shown in Figure 1-15 [5]. This definition would also
include viruses [6], which microbiology texts traditionally discuss along with living
organisms.

Figure 1-15 Some examples of the types of microbes present in the

environment
Many different organisms fall under the definition of microorganisms. Shown here are:
A, the bacterium Escherichia coli; B, a photosynthetic cyanobacterium; C, a fungus; D,
Ebola virus; E, the protozoan malaria parasite. (Sources: B, Mike Clayton; C-E, CDC).
Note that the scale on each of these pictures is different.

Microbiology also involves a collection of techniques to study and manipulate these

small creatures. Because of their size, special instruments and methods had to be
developed to allow the performance of interpretable experiments on microorganisms.
These methods are not restricted to microbes alone, but have also found utility in
working with populations of cells from higher organisms.

With apologies to other small organisms, this book will mostly focus on bacteria (which
we will also call microorganisms or microbes) and their impact on the rest of the
biosphere [7]. This can be weakly justified by the fact that bacteria have a major impact
on the world around us and, because of their perceived importance, more research and
knowledge has been accumulated about them.

Microorganisms are everywhere, but why are they worth learning about? The short
answer is that they affect your life in many different ways. Before we begin our study of
these creatures, we will first take a tour of some of their important habitats and point out
why your existence depends upon them. We will then briefly explore the history of
microbiology.

1 - 2 Microbes have a large impact on human health

  Microbes cause many infectious diseases.
 Vaccines, antibiotics, and many other advances have lessened the impact of
infectious disease in the developed world, but infectious disease in developing
countries is high.
 New illnesses caused by microorganisms continue to emerge and known
pathogens are becoming resistant to treatment.

If you ask the average person how microbes (or germs) impact their lives, they would
immediately think of disease. This is not a silly view, as Figure 1-16 [8] shows a
number of important pathogens.

Figure 1-16 Some important pathogens

Many microbes cause disease in humans. Depicted here are several pathogens that cause
important illnesses. A, Influenza virus; B, West Nile Virus; C, Staphylococcus aureus;
D, Streptococcus pneumoniae. (Sources: A, Dr. Erskine/L. Palmer/ Dr. M. L. Martin; B,
Cynthia Goldsmith; C, Janice Carr/ Jeff Hageman, M.H.S.; D, Dr. Mike Miller; all
individuals are at the CDC.)

While death from infectious disease in the U.S. has been greatly diminished, infection
rates in developing nations remain unacceptably high. "Ancient" diseases continue to be
a problem where nutrition and sanitation are poor, and emerging diseases such as
Acquired Immunodeficiency Syndrome (AIDS) are even more dangerous for such
populations. The Centers for Disease Control and Prevention (the U.S. government
agency charged with protecting human health and safety) estimate that about 9% of
adults between the ages of 18-49 in Sub-Saharan Africa are infected with HIV. Yet as
you can see in Figure 1-13 [9], AIDS is only one of a number of new diseases that have
emerged. Many of the new diseases are viral in nature, making them notoriously
difficult to treat and they have no known cure. Influenza and pneumonia are leading
killers of the elderly even in the U. S. and other developed nations. Even the common
cold causes illness and misery for almost everyone and drains the productivity of all
nations.

Disease due to food-borne pathogens also remain a problem, largely because of

consumption of improperly processed or stored foods. Understanding the sources of
contamination and developing ways to limit the growth of pathogens in food is the job
of food microbiologists.

Figure 1-13 Disease-causing microbes and infectious diseases recognized

since 1973

Year Microbe/disease Type Health problem

1973 Rotavirus Virus Major cause of infantile diarrhea worldwide
1975 Parvovirus B19 Virus Severe anemia
1976 Cryptosporidium parvum Parasite Acute and chronic diarrhea
Ebola hemorrhagic fever/uncontrolled
1977 Ebola Virus
bleeding and kidney failure
1977 Legionella pneumophila Bacteria Legionnaire's disease
1977 Hanta virus Virus Hemorrhagic fever
1977 Campylobacter jejuni Bacteria Short-term diarrhea
Human T-lymphotropic virus T-cell lymphoma-leukemial cancer of the
1980 Virus
I (HTLV-I) blood
Toxic strains of Staphyloccus
1981 Bacteria Toxic shock syndrome
aureus
Hemorrhagic colitis; hemolytic uremic
1982 Escherichia coli O157:H7 Bacteria
syndrome
1982 HTLV-II Virus Hairy cell leukemia
1982 Borrelia burgdorferi Bacteria Lyme disease
Human immunodeficiency Acquired immune deficiency syndrome
1983 Virus
virus (HIV) (AIDS)
1983 Helicobacter pylori Bacteria Peptic ulcer disease
1985 Entercytozoon bieneusi Parasite Persistent diarrhea
1986 Cyclospora cayetanensis Parasite Persistent diarrhea
Human herpesvirus-6 (HHV-
1988 Virus Roseola subitum/skin rash
6)
1988 Hepatitis E Virus Liver infection; epidemic hepatitis
1989 Ehrlichia chaffeensis Bacteria Human ehrlichiosis/influenza-like infection
1989 Hepatitis C Virus Chronic liver infection
1991 Guanarito virus Virus Venezuelan hemorrhagic fever
1991 Encephalitozoon hellem Parasite Conjunctivitis
Atypical babesiosis/infection with fever,
1991 New species of Babesia Parasite
chills and fatigue
Catch scratch disease/bacillary
1992 Bartonella henselae Bacteria
angionmatosis
1993 sin nombre virus Virus Adult respiratory distress syndrome
1993 Encephalitozoon cuniculi Virus Infection with fever, chills and fatigue
1994 Sabia virus Virus Brazilian hemorrhagic fever
 Associated with Kaposi sarcoma in AIDS
1995 HHV-8 Virus
patients

Source: WHO, The World Health Report 1996: 112

New infections continually appear. Having an available food source to grow on

(humans) inevitably results in a microorganism that will take advantage. Some of these
feeders will interfere with our own well being, causing disease.

Surprisingly, many diseases that were previously thought to have only behavioral or
genetic components have been found to involve microorganisms. The clearest case is
that of ulcers, which was long thought to be caused by stress and poor diet. However the
causative agent is actually a bacterium, Helicobacter pylori, and many ulcers can be
cured with appropriate antibiotics. Work on other non-infectious diseases such as heart
disease, stroke and some autoimmune diseases also suggest a microbial component that
triggers the illness.

Finally, some pathogenic microbes that had been "controlled" through the use of
antibiotics are beginning to develop drug resistance and therefore reemerge as serious
threats in the industrialized world as well as developing nations. Tuberculosis is an
illness that was on the decline until the middle 80's. It has recently become more of a
problem, partly due to drug resistance and partly due to a higher population of
immunosuppressed individuals from the AIDS epidemic. Staphylococcus aureus strains
are emerging that are resistant to many of the antibiotics that were previously effective
against them. These staph infections are of great concern in hospital settings around the
world. Understanding both familiar killers and new pathogens will require an
understanding of their biology, and thus an understanding of the field of microbiology.

1 - 3 Microbes are often helpful, not harmful

 Microbes form important mutualistic relationships with all sorts of organisms.

Many of these relationships are important from a human perspective.

From the beginning of microbiology, significant resources have been spent to

understand and fight disease-causing microorganisms. You may be surprised to learn
that only a small fraction of microbes are involved in disease; many other microbes
actually enhance our well being. The harmless microbes that live in our intestines and
on our skin actually help us fight off disease. They actively antagonize other bacteria
and take up space, preventing potential pathogens from gaining a foothold on our
bodies. The microbial community in humans not only protects us from disease, but also
provides needed vitamins, such as B12. We have entire communities of microorganisms
in our digestive systems that contribute to our overall health. In fact, like all other large
organisms, humans are actually consortia of different organisms - there are more non-
human cells in and on our bodies than there are human cells!

Human health and nutrition also depends on healthy farm animals. Cows, sheep and
other ruminant animals utilize their microbial associates to degrade plant material into
useful nutrients. Figure 1-1 [10] shows the cow, one example of a ruminant animal.
Without these bacteria inside ruminants, growth on plant material would be impossible.
Figure 1-1 The cow as an example of a ruminant animal

In contrast to humans, ruminant animals have a complex system of stomachs that harbor
large numbers of microorganisms. These microbes degrade the tough plant material
eaten by the animal into usable nutrients. Without the assistance of the microbes,
ruminant animals would not be able to digest the food they eat. (Source: Keith Weller,
USDA.)

Commercial crops are also central to human prosperity, and much of agriculture
depends upon the activities of microbes. For example, an entire group of plants, the
legumes, forms a cooperative relationship with certain bacteria. These bacteria convert
nitrogen gas to ammonia for the plant, an important nutrient that is often limiting in the
environment. Figure 1-17 [11] shows a leguminous plant and the special structures on
the roots that house these helpful bacteria. Microbes also serve as small factories,
producing valuable products such as cheese, yogurt, beer, wine, organic acids and many
other items. In conclusion, while it is less apparent to us, the positive role of microbes in
human health is at least as important as the negative impact of pathogens.

Figure 1-17 A leguminous plants

Nitrogen-fixing bacteria form special structures, called nodules, on the roots of
leguminous plants. In the picture, peas are shown that have either been exposed to
bacteria (left) or not (right). The small bumps on the roots on the left are the nodules
and contain millions of bacteria actively fixing N2 for the plant's use. Though not
obvious from this figure, the plants on the left are more robust because of that nitrogen.
(Source: E. B. Fred, et al., 1921. Soil Science 11:479-491.)

1 - 4 Microbes have profound effects on the environment

 Microbes make up the major portion of the biomass present on the Earth.
Therefore, the nutrients they eat and the products they form greatly influence the
environment.
 Cyanobacteria and algae in the oceans are responsible for most photosynthesis
and are a major sink for carbon dioxide, a greenhouse gas.
 Microbes release nutrients from dead organisms, making them available to the
rest of the ecosystem.
 Some microbes play a role in the production of energy, while other microbes
interfere with energy production.

Whether measured by the number of organisms or by total mass, the vast majority of
life on this planet is microscopic. These teaming multitudes profoundly influence the
make-up and character of the environment in which we live. Presently, we know very
little about the microbes that live in the world around us because less than 2 % of them
can be grown in the laboratory. Understanding which microbes are in each ecological
niche and what they are doing there is critical for our understanding of the world.
Figure 1-2 [12] shows some examples of environments where microbes are present

Microbes are the major actors in the synthesis and degradation of all sorts of important
molecules in environments. Cyanobacteria [13] and algae in the oceans are responsible
for the majority of photosynthesis on Earth. They are the ultimate source of food for
most ocean creatures (including whales) and replenish the world's oxygen supply.
Cyanobacteria also use carbon dioxide to synthesize all of their biological molecules
and thus remove it from the atmosphere. Since carbon dioxide is a major greenhouse
gas, its removal by cyanobacteria affects the global carbon dioxide balance and may be
an important mitigating factor in global warming.

Figure 1-2 A sampling of the different types of environments where

microbes flourish

Microbes are capable of growing in a wide variety of environments. Bacteria will grow
in frigid glaciers to boiling volcanic springs, dry sands to the open ocean. Figure
courtesy of Kelsea Jewell

In all habitats, microorganisms make nutrients available for the future growth of other
living things by degrading dead organisms. Microbes are also essential in treating the
large volume of sewage and wastewater produced by metropolitan areas, recycling it
into clean water that can be safely discharged into the environment. Less helpfully
(from the view of most humans), termites contain microorganisms in their guts that
assist in the digestion of wood, allowing the termites to extract nutrients from what
would otherwise be indigestible. Understanding of these systems helps us to manage
them responsibly and as we learn more we will become ever more effective stewards.

Energy is essential for our industrial society and microbes are important players in its
production. A significant portion of natural gas comes from the past action of
methanogen [14]s (methane-producing bacteria). Numerous bacteria are also capable of
rapidly degrading oil in the presence of air and special precautions have to be taken
during the drilling, transport and storage of oil to minimize their impact. In the future,
microbes may find utility in the direct production of energy. For example, many
landfills and sewage treatment plants capture the methane produced by methanogens to
power turbines that produce electricity. Excess grain, crop waste and animal waste can
be used as nutrients for microbes that ferment this biomass [15] into methanol or
ethanol. These biofuel [16]s are presently added to gasoline and thus decreasing
pollution. They may one day power fuel cells in our cars, causing little pollution and
having water as their only emission.

Finally, we are increasingly taking advantage of the versatile appetite of bacteria to

clean up environments that we have contaminated with crude oil, polychlorinated
biphenyls (PCBs) and many other industrial wastes. This process is termed
bioremediation [17] and is a cheap and increasingly effective way of cleaning up
pollution. Figure 1-18 [18] shows before and after photographs of the clean up of the
Exxon Valdez oil spill in Prince William Sound.

Figure 1-18 The Exxon Valdez oil spill

Microorganisms played an important role in removing many of the pollutants released

during the Exxon Valdez oil spill in Prince William Sound. Interestingly, microbes were
not added to the site, but the clean-up relied on bacteria from that environment. A
nutrient solution was sprayed onto the oil to encourage the growth of oil-degrading
microbes. Though this was one of the more successful methods used to clean up the oil,
but no treatment removed all of the pollutants. (Source:
http://www.battelle.org/environment/exxon-valdez.stm.)

1 - 5 Studying microbes helps us to understand the world around us

 Microbes are useful tools in research because of their rapid life cycle, their
simple growth requirements, and their small size.
 Due to this simplicity, microbes have been essential in understanding core
questions in biology.
 Attempts to classify microorganisms have lead to a classification system that
divides all organisms into three domains of life: Archaea, Bacteria, and Eukarya.
 Microbes provide tools for use in molecular biology. These tools have allowed
scientists to make rapid progress in investigating many types of microorganisms.

Microorganisms used in research have many useful properties. They grow on simple,
cheap medium and often give rise to large populations in a matter of 24 hours. It is easy
to isolate their genomic material, manipulate it in the test tube and then place it back
into the microbe. Due to their large populations it is possible to identify rare events and
then, with the use of powerful selective techniques, isolate interesting bacterial cells and
study them. These advantages have made it possible to test hypotheses rapidly. Using
microbes scientists have expanded our knowledge about life. Below are a few examples.

Microorganisms have been indispensable instruments for unlocking the secrets of life.
The molecular basis of heredity and how this is expressed as proteins was described
through work on microorganisms. For an in-depth discussion on the molecular basis of
heredity see the chapter on the Central Dogma. Due to the similarity of life at the
molecular level, this understanding has helped us to learn about all organisms, including
ourselves.

Some prokaryotes are capable of growing under unimaginably harsh conditions and
define the extreme limits of where life can exist. Some species have been found
growing at near 100 °C in hot springs and well above that temperature near deep-sea
ocean vents. Figure 1-19 [19] depicts such a deep sea ocean vent. Others make their
living at near 0 °C in freshwater lakes that are buried under the ice of Antarctica. The
ability of microbes to live under such extreme conditions is forcing scientists to rethink
the requirements necessary to support life. Many now believe it is entirely possible that
Jupiter's moon Europa may harbor living communities in waters deep below its icy
crust. What may the rest of the universe hold?

Figure 1-19 A deep sea ocean vent

Ocean vents are common in areas of the sea floor that have volcanic activity. Water
seeps into cracks on the floor and encounters magma. The water absorbs inorganic
nutrients from the magma and is heated. The superheated water then flows out of the
magma, sometimes quite forcefully, back into the ocean. This hot water contacts the
cold ocean water, causing it to cool and release many of its inorganic contents. This
cloud of inorganic compounds is highly reduced and can serve as a source of energy for
microorganisms. These microbes serve as a primary producer for an entire food chain.
The picture shows one type of ocean vent called a black smoker. Water coming out of
the vent can be >300°C. Figure courtesy of Woods Hole Oceanic Institute.
Until recently, while we could study specific types of bacteria, we lacked a cohesive
classification system, so that we could not readily predict the properties of one species
based on the known properties of others. Visual appearance, which is the basis for
classification of large organisms, simply does not work with many microbes because
there are few distinguishing characteristics for comparison between species even under
the microscope. However, analysis of their genetic material in the past 20 years has
allowed such classification and spawned a revolution in our thinking about the
evolution of bacteria and all other species. The emergence of a new system organizing
life on Earth into three domains is attributable to this pioneering work with
microorganisms.

The fruits of basic research on microbes have been used by scientists to understand
microbial activity and therefore to shape our modern world. Human proteins, especially
hormones like insulin and human growth factor, are now produced in bacteria using
genetic engineering. Our understanding of the immune system was developed using
microbes as tools. Microorganisms also play a role in treating disease and keeping
people healthy. Many of the drugs available to treat infectious disease originate from
bacteria and fungi.

Lastly, microbes have informed us about our world through the tools they provide for
molecular biology. Enzymes purified from bacterial strains are useful as tools to
perform many types of analyses. Such analyses allow us to determine the complete
genome sequence of almost any organism and manipulate that DNA in useful ways. We
now know the entire sequence of the human genome, with the exception of regions of
repetitive DNA, and this will hopefully lead to medical practices and treatments that
improve health. We also know the entire genome sequences of hundreds of microbes,
including those of many important pathogens. Analysis of these data will eventually
lead to an understanding of the function of critical enzymes in these microbes and the
development of tailor-made drugs to stop them. The tools of molecular biology will also
affect agriculture. For example, we now know the complete genome sequence of the
plant Arabidopsis (a close relative of broccoli and cauliflower). This opens a new
avenue to a better understanding of all plants and hopefully improvements in important
crops.

Microbes have a profound impact on every facet of human life and everything around
us. Pathogens harm us, yet other microbes protect us. Some microbes are pivotal in the
growth of food crops, but others can kill the plants or spoil the produce. Bacteria and
fungi eliminate the wastes produced in the environment, but also degrade things we
would rather preserve. Clearly they affect many things we find important as humans. In
the remainder of this chapter we take a look at how scientists came to be interested in
microbes and follow a few important developments in the history of microbiology.

1 - 6 The history of microbiology is a web of discoveries

 Science is interdependent and new discoveries depend upon earlier contributions

from many other scientists.

Before we begin the adventure that we call learning microbiology (it can be thought of
as an adventure! Really!) a look at the history of microbiology will help you to
understand the contributions of those who have come before. This perspective will
hopefully give you an appreciation of their efforts and put the body of knowledge we
will examine in the context of history. Keep in mind that microbiology is a relatively
young science. It was only about 140 years ago that it became possible to seriously
study microorganisms in the laboratory, with most of our understanding of microbes
coming in the last 60 years.

The history of microbiology, like all human history, is not a catalog of linear progress,
but is more of an interweaving of the careers of bright individuals and their insights.
Each new discovery relied on previous ones and in turn spawned further inquiry. A web
of interdependent concepts evolved over time through the work of scientists in many
related disciplines and nations. Often the research of one individual impacted the efforts
of another studying a completely different problem. Keep this in mind as you look at
this history.

Below we present several journeys through this web, mentioning some individuals who
were particularly important in the progression. This history reflects our view of
important events of the past, but is by no means comprehensive. We will first look at
the development of the techniques for handling microorganisms, since everything else
in microbiology depends upon these procedures. Next, we will examine how these
techniques helped to settle an old debate, the question of spontaneous generation. Then,
we will look at the history of infectious disease. The science of microbiology had its
most significant early impact on human health, uncovering the cause of the major killers
of the day, and then methods to treat them. As microbiology matured, scientists began
to look at what non-pathogenic microbes were doing in the environment and we will
look a bit at the history of general microbiology. Finally, the chapter will end with an
examination of the events that lead to the understanding of life at the molecular level
and the profound impact this has had on microbiology and on society in general.

1 - 7 Microscopes allowed the discovery of microbes

 Microbes were first seriously described in the 17th century by Robert Hooke and
Anton van Leeuwenhoek using simple microscopes.
 Ferdinand Cohn continued this work many years later, making a first systematic
attempt at classifying them.

For years the existence of microorganisms was suspected, but could not be proven,
since bacteria were too small to be seen with the naked eye. It took the microscope to
expose their tiny world and that instrument has been linked to microbiology ever since.
In 1664, Robert Hooke devised a compound microscope and used it to observe fleas,
sponges, bird feathers, plants and molds, among other items. His work was published in
Micrographia and became a popular and widely read book at the time.

Several years later Anton van Leeuwenhoek, a fabric merchant and amateur scientist (or
"natural philosopher" as such people called themselves), became very adept at grinding
glass lenses to make telescopes and microscopes. While crude by modern standards, his
were a technical marvel for the time, able to magnify samples greater than 200-fold - a
reproduction of a Leeuwenhoek microscope is shown in Figure 1-3 [20]. They also
produced clearer images than the compound microscopes of the time. By peering
through his microscope, Leeuwenhoek observed tiny organisms or "wee animacules" as
he called them. He spent months looking at every kind of sample he could find and
eventually submitted his observations in a letter to the Royal Society of London,
causing a sensation. Hooke was asked to confirm the findings of Leeuwenhoek and his
affirmative assessment garnered them wide acceptance. Surprisingly the work of these
two scientists was not followed up for almost 200 years. Human societies had neither
the technical prowess nor the inclination to develop the science of microorganisms. It
was not until the rise of the industrial revolution that governments and people dedicated
the financial and physical resources to understand these small inhabitants of our world.

Figure 1-3 The Van Leeuwenhoek microscope

The left panel shows a replica of a Leeuwenhoek microscope . The photomicrographs in

the center and right were taken in the early 20th century through one of Leeuwenhoek's
microscope. (Source: The Leeuwenhoek Letter. Society of American Bacteriologists.
Baltimore. 1937.)

With the development of better microscopes in the 19th century, scientists returned to an
examination of microorganisms. After finishing his education, Ferdinand Julius Cohn
was able to convince his father to lay down the large sum necessary to purchase a
microscope for him, one better than that available at the University in Breslau, then part
of Germany. He used it to carefully examine the world of the microbe and made many
observations of eukaryotic microorganisms and bacteria. His landmark papers on the
cycling of elements in nature was published in Ueber Bakterien in 1872 and a microbial
classification scheme including descriptions of Bacillus were published in the first
volume of a journal he founded, Beitraege zur Biologie der Planzen. Cohn's work with
microscopes popularized their use in microbiology. This and his other work inspired
many other scientists to examine microbes. Cohn's encouragement of Robert Koch, a
German physician by training, began the field of medical microbiology.

1 - 8 Robert Koch developed many microbiological techniques

 Many of Koch's methods for cultivating microbes are still used today.

Robert Koch, pictured in Figure 1-4 [21], searched for the causes of many diseases.
Through these investigations he and his laboratory developed many classic microbial
techniques. He used adaptations of the staining methods of Carl Weigert to begin the
process of distinguishing microbes and identifying pathogens. His lab was the first to
isolate a disease-causing organism.

Figure 1-4 Robert Koch

The German Physician Robert Koch. Drawing by Tammi Henke

A major contribution to bacterial techniques was the development of methods using

solid medium. For example LB medium or minimal medium for the cultivation of
bacteria. Koch was convinced that microbes caused some diseases. However, to test this
idea, he needed to isolate the causative agent. Almost all samples from diseased animals
or any natural surface contained many different microbes and it was impossible to tell
which one was the problem. A method was needed to separate these different bacteria.
The most common method of isolation was to continually dilute a sample in liquid broth
in hopes that at high enough dilution, only one type of microbe would be found. A
problem with this method is that only the most populous microbe would be isolated, but
that might not be one causing the disease. There were other technical problems as well
with such a liquid-based system, so a solid medium would seem to provide distinct
advantages. Koch had tried gelatin for these experiments with unsatisfactory results.
Building on the work of Brefeld and Schroeter, Koch used potato slices as a solid
medium and observed that a boiled potato left in the open air would develop tiny
circular raised spots.

Examination of these spots revealed they were made up of microorganisms and each
spot had just one type of microbe in it. He realized that these colonies were pure
cultures of bacteria and probably arose from a single species of microbe from the air
that landed on the potato. By boiling a potato, slicing it with a hot knife and keeping it
in a sterile container with a lid, Koch could keep the potato sterile. But if a sample from
a disease animal was smeared across the potato, colonies arose, each being pure isolates
from the animal. By then testing these isolates in animals, Koch was able to isolate the
cause of anthrax, Bacillus anthracis.

Potatoes failed to support the growth of many microorganisms and Koch and his
laboratory were constantly frustrated by the lack of a good solid medium. Walter Hesse
joined Koch's laboratory to do studies on air quality, showing a remarkable attention to
detail and patience in his work. His wife, Angelina Fannie Hesse along with raising
their three sons, also would assist her husband with his research in the laboratory.
Walter was attempting to do his air quality experiments using medium containing
gelatin as the solidifying agent. In the summertime, temperatures would often rise above
the melting point of gelatin. In addition, microbes would often grow in the cultures that
were capable of degrading gelatin and in both cases this would cause liquefaction of the
medium, ruining the experiments. One day while eating lunch, the frustrated scientist
asked Lina (as she was called) why her jellies and puddings stayed solid even in the hot
summer temperatures. She told him about agar-agar, a heat resistant gelling agent that
she had learned about while growing up in New York from a Dutch neighbor who had
emigrated from Java.

Development of the new agent by Angelina and Walter led to a resounding success.
Few microbes are able to degrade agar and it melts at 100 °C yet remains molten at
temperatures above 45 °C. This allows the mixing of the agar with heat-sensitive
nutrients and microbes. After solidification, it does not melt until a temperature of 100
°C is again attained, facilitating the easy cultivation of pathogens. It can also be stored
for long periods of time, allowing the cultivation of slow-growing microorganisms. Any
type of broth can be mixed with agar, giving great flexibility in the kinds of medium
that can be made. Thus, many more types of microbes could be cultivated.

Koch's laboratory also developed methods of pure culture maintenance and aseptic
technique. Aseptic technique involves the manipulation of pure cultures in a manner
that prevents their contamination by outside microorganisms. Equally important, aseptic
technique prevents their spread into the environment. Remember that Koch was
studying some of the most devastating microbial pathogens of the period, and their
release could potentially cause disease in the scientists working on them. These
procedures were also absolutely critical because they allowed careful study of pure
microorganisms, making it possible to identify the role of each microbe in a given
situation.

Another problem in the cultivation of microbes was solved by Julius Petri while
working in Koch's laboratory. Solid medium was poured on glass plates and allowed to
spread and harden. Once cooled it allowed a solid surface for streaking. However,
creation of these plates required great care since exposure to the air (and the microbes in
it) often lead to contamination. In addition, to prevent contamination of plates during
incubation, a cumbersome bell jar was used. If one wanted to view samples, the plate
had to be removed from the jar, further exposing it to unwanted microbes in the air. In
1887 Petri developed shallow glass dishes, with one having a slightly larger diameter
than the other. Medium is poured into the smaller dish and the larger one serves as a
cover. This simple device solved all of the above problems and took on the name of its
inventor, the petri plate.
These same techniques are essential in studying all microorganisms. Collectively the
above techniques have been used to isolate and identify thousands of different
microorganisms. As a testament to the significance of their achievement, these
techniques are practiced with remarkably little change in every laboratory that works
with microorganisms today. Figure 1-27 [22] lists some of the early advances that
helped to develop the practice of microbiology.

Figure 1-27 The development of early techniques in microbiology.

Year Event
Robert Hooke is the first to use a microscope to describe the fruiting structures of
1664 molds. He also coined the term cell when using a microscope to look at cork, as
the dead plant material in cork reminded him of a jail cell.
Anton van Leeuwenhoek, a Dutch tradesman and skilled lens maker, is the first to
1673
describe microbes in detail.
Ferdinand Julius Cohn publishes landmark paper on bacteria and the cycling of
1872
elements. In it is an early classification scheme that uses the name Bacillus.
Oscar Brefeld reports the growth of fungal colonies from single spores on gelatin
1872 and the German botanist Joseph Schroeter grows pigmented bacterial colonies on
slices of potato.
Robert Koch develops methods for staining bacteria, photographing, and preparing
1877
permanent visual records on slides.
Koch develops solid culture media and the methods for obtaining pure cultures of
1881
bacteria.
Angelina Fannie and Walther Hesse in Koch's laboratory develop the use of agar
1882
as a support medium for solid culture.
Hans Christian Gram develops a dye system for identifying bacteria [the Gram
1884
stain].
1887 First report of the petri plate by Julius R. Petri.
M. H. McCrady establishes a quantitative approach for analyzing water samples
1915
using the most probable number, multiple-tube fermentation test.

1 - 9 Spontaneous generation was an attractive theory to many people,

but was ultimately disproven.

 For many centuries many people believed in the concept of spontaneous

generation, the creation of life from organic matter.
 Francesco Redi disproved spontaneous generation for large organisms by
showing that maggots arose from meat only when flies laid eggs in the meat.
 Spontaneous generation for small organisms again gained favor when John
Needham showed that if a broth was boiled (presumed to kill all life) and then
allowed to sit in the open air, it became cloudy.
 Louis Pasteur ended the debate with his famous swan-neck flask experiment,
which allowed air to contact the broth. Microbes present in the dust were not
able to navigate the tortuous bends in the neck of the flask.

Spontaneous generation is the hypothesis that some vital force contained in or given to
organic matter can create living organisms from inanimate objects. Spontaneous
generation was a widely held belief throughout the middle ages and into the latter half
of the 19th century. In fact, some people still believe in it today. The idea was attractive
because it meshed nicely with the prevailing religious views of how God created the
universe. There was a strong bias to legitimize the idea because this vital force was
considered a strong proof of God's presence in the world. Many recipes and experiments
were offered in proof. To create mice, a recipe called for dirty underwear and wheat
grain to be mixed in a bucket and left open outside. In 21 days or less, you would have
mice. The real cause may seem obvious from a modern perspective, but to the
proponents of this idea, the mice spontaneously arose from the wheat kernels.

Another often-used example was the generation of maggots from meat that was left in
the open. The failing here was revealed by Francesco Redi in 1668 with a classic
experiment. Redi suspected that flies landing on the meat laid eggs that eventually grew
into maggots. To test this idea he devised the experiment shown in Figure 1-20 [23].
Here he used three pieces of meat. One piece of meat was placed under a piece of paper.
The flies could not lay eggs onto the meat and no maggots developed. The second piece
was left in the open air, resulting in maggots. In the final test, a third piece of meat was
overlaid with cheesecloth. The flies were able to lay the eggs into the cheesecloth and
when this was removed no maggots developed. However, if the cheesecloth containing
the eggs was placed on a fresh piece of meat, maggots developed, showing it was the
eggs that "caused" flies and not spontaneous generation. This helped to end the debate
about spontaneous generation for large organisms. However, spontaneous generation
was so seductive a concept that even Redi believed it was possible in other
circumstances.

Figure 1-20 The Redi experiment

The concept and the debate were revived in 1745 by the experiments of John Needham.
It was known at the time that heat was lethal to living organisms. Needham theorized
that if he took chicken broth and heated it, all living things in it would die. After heating
some broth, he let a flask cool and sit at a constant temperature. The development of a
thick turbid solution of microorganisms in the flask was strong proof to Needham of the
existence of spontaneous generation. Lazzaro Spallanzani later repeated the experiments
of Needham, but removed air from the flask, suspecting that the air was providing a
source of contamination. No growth occurred in Spallanzani's flasks and he took this as
evidence that Needham was wrong. Proponents of spontaneous generation discounted
the experiment by asserting that air was required for the vital force to work.

It was not until almost 100 years later that the great French chemist Louis Pasteur,
pictured in Figure 1-5 [24], put the debate to rest. He first showed that the air is full of
microorganisms by passing air through gun cotton filters. The filter trapped tiny
particles floating in the air. By dissolving the cotton with a mixture of ether and alcohol,
the particles were released and then settled to the bottom of the liquid. Inspection of this
material revealed numerous microbes that resembled the types of bacteria often found in
putrefying media. Pasteur realized that if these bacteria were present in the air then they
would likely land on and contaminate any material exposed to it.

Figure 1-5 Louis Pasteur

Pasteur then entered a contest sponsored by The French Academy of Sciences to

disprove the theory of spontaneous generation. Similar to Spallanzani's experiments,
Pasteur experiment, pictured in Figure 1-6 [25], used heat to kill the microbes, but left
the end of the flask open to the air. In a simple, but brilliant modification, the neck of
the flask was heated to melting and drawn out into a long S-shaped curve, preventing
the dust particles and their load of microbes from ever reaching the flask. After
prolonged incubation the flasks remained free of life and ended the debate for most
scientists.

Figure 1-6 The swan neck flask experiment

A final footnote on the topic was added when John Tyndall showed the existence of
heat-resistant spores in many materials. Boiling does not kill these spores and their
presence in chicken broth, as well as many other materials, explains the results of
Needham's experiments.

While this debate may seem silly from a modern perspective, remember that the
scientists of the time had little knowledge of microorganisms. Koch would not isolate
microbes until 1881. The proponents of spontaneous generation were neither sloppy
experimenters nor stupid. They did careful experiments and interpreted them with their
own biases. Detractors of the theory of spontaneous generation were just as guilty of
bias, but in the opposite direction. In fact, it is somewhat surprising that Pasteur and
Spallanzoni did not get growth in their cultures, since the sterilization conditions they
used would often not kill endospores. Luck certainly played a role. It is important keep
in mind that the discipline of science is performed by humans with all the fallibility and
bias inherent in the species. Only the self-correcting nature of the practice reduces the
impact of these biases on generally held theories. Spontaneous generation was a severe
test of scientific experimentation, because it was such a seductive and widely held
belief. Yet, even spontaneous generation was overthrown when the weight of careful
experimentation argued against it. Figure 1-21 [26] lists important events in the
spontaneous generation debate.

Figure 1-21 Events in spontaneous generation

Year Event
Francesco Redi attacks spontaneous generation and disproves it for large
1668
organisms
John Needham adds chick broth to a flask and boils it, lets it cool and waits.
1745
Microbes grow and he proposes it as an example of spontaneous generation.
Lazzaro Spallanzani repeats Needham's experiment, but removes all the air from
1768
the flask. No growth occurs.
1859 Louis Pasteur's swan-neck flasks show that spontaneous generation does not occur.
Thomas H. Huxley gives his "Biogenesis and Abiogenesis" lecture. The speech
1870 offered powerful support for Pasteur's claim to have experimentally disproved
spontaneous generation.
John Tyndall publishes his method for fractional sterilization, showing the
1877
existence of heat-resistant bacterial spores.

1 - 10 Microbes are discovered to cause disease

 Ignaz Semmelweis showed that child-bed fever was spread by physicians and
could be prevented by careful hand washing with chloride of lime.
 Louis Pasteur, while working on sour wine, discovered that unwanted microbes
were infecting the wine. He correctly deduced that infectious disease was caused
by similar infections with harmful microbes.
 Robert Koch was the first to isolate a disease-causing microbe, Bacillus
antrhacis. In the process he developed techniques and standard protocols for
defining the cause of a disease.

It was long suspected that living things were the agents of disease. In volume 6 of his
epic poem De Rerum Natural (On the Nature of the Universe) written sometime around
50 B.C., Titus Lucretius Carus speculates about invisible atoms causing disease. This
was only one idea among many and some thought that an imbalance in humors caused
illness, while others felt that supernatural forces were at work. The prevailing theory
held by most doctors of the 19th century was that chemical toxins were carried from an
ill patient to others, causing them to contract the same malady. The bacteria that were
known to be present were seen as a symptom of the disease and not its cause.

Ignaz Semmelweis, pictured in Figure 1-7 [27], a Hungarian physician working in

Vienna, made the first breakthrough in the true nature of disease. He realized that
asepsis in obstetrical wards could prevent the transmission of childbirth fever from
patient to patient. He therefore instigated a policy for all attending physicians to wash
their hands with chloride of lime (a mixture of calcium chloride hypochlorite,
CaCl(OCl); calcium hypochlorite, Ca(OCl)2; and calcium chloride, CaCl2) between
patients. This innovation dropped the mortality rate from 18% to 2.4%.

Figure 1-7 Ignaz Semmelweis

Ignaz became a vigorous proponent of his ideas, but the Hungarian doctor's efforts were
opposed by many who could not accept that physicians themselves could be responsible
for spreading bacterial infection. Ridicule of his idea caused him to move from Vienna
to Pest, Hungary and ultimately played a role in a nervous breakdown. Ironically
Semmelweis died from an infection that he contracted during a surgery he performed,
while recovering from his nervous breakdown.

Before his death he published his ideas in a paper The Cause, Concept, and Prophylaxis
of Childbed Fever in 1861. Although the poor writing of the paper contributed to the
obscurity of his ideas, the work was ignored for 17 years, which raises an interesting
point about the culture of science. Radical ideas, even those that are correct and can
save lives, are sometimes ignored. It takes time to overcome the dogma of the day. The
personalities involved and the negative light it might throw on past practices play a
large role in the rate of acceptance of a new idea.

In a seemingly unrelated event, Louis Pasteur found something interesting while

working on wine souring, a problem where wine fermentations produce a sour taste and
very little alcohol. Fermentation of alcoholic beverages was thought to be a simple
chemical reaction. Heating, common in most beverage preparations, was thought to
cause the breakdown of sugar into alcohol. Pasteur was asked to help out the wine
industry in France because wine souring was pushing it close to ruin. His work on wine-
making revealed that the process of converting sugar to alcohol is actually performed by
various yeast strains. He then showed that the wine was going bad because a
contaminating microbe was generating lactic acid instead of alcohol from the sugar.
This idea was controversial, but gained credibility when Pasteur solved the problem by
heating the wine and killing the contaminant. The heating process was named
pasteurization in his honor and is still widely used today. In a brilliant step of
generalization, Pasteur realized that souring of wine and infectious disease shared a
common thread in that they both might involve infection by a microorganism. His
suggestion that microbes cause disease became known as the germ theory of disease.

Joseph Lister became aware of Semmelweis's work and together with Pasteur realized
the true nature of disease. He then recognized that he could use this idea to help his
surgery patients. At this time, major injuries, broken bones or surgery would often result
in infection of the damaged area, sometimes leading to amputation or death. Lister
found he could greatly reduce the number of microorganisms on wounds and incisions
by using bandages treated with phenic acid, a compound that killed microorganisms
(phenic acid, now known as phenol, is the active ingredient in Listerine). During
surgery he began the practice of spraying the wound with a fine mist of phenic acid to
kill microbes. These practices greatly reduced the rate of infection and mortality of
surgery patients, lending further credence to the germ theory of disease.

In 1876 Robert Koch provided definitive proof of the germ theory by isolating the cause
of anthrax and showing it to be a bacterium. From this came the development of Koch's
Postulates, a set of rules for the assignment of a microbe as the cause of a disease:

1. The specific organism should be shown to be present in all cases of animals

suffering from a specific disease, but should not be found in healthy animals.
2. The specific microorganism should be isolated from the diseased animal and
grown in pure culture on artificial laboratory media.
3. This freshly isolated microorganism, when inoculated into a healthy non-
immune laboratory animal, should cause the same disease seen in the original
animal.
4. The microorganism should be reisolated in pure culture from the experimental
infection.

The postulates are Koch's most famous contribution to science and it is a testament to
the utility of these postulates that they are stilled used today to discover the cause of
new emerging diseases. Koch went on to apply these principles in the study of many
other diseases including tuberculosis, cholera and sleeping sickness. It should be
pointed out that Koch's postulates cannot be applied to all diseases. For example if a
disease-causing microbe has humans as its sole host and has a significant possibility of
causing death, it would be unethical to apply this microbe to test humans as dictated by
postulate 3. Also, it is not always possible to obtain a disease-causing microbe in pure
culture.

While attacking the problem of disease, Koch developed the tools for obtaining pure
cultures. Advances in science often come from innovations in the available technology.
Robert Koch was an important microbiologist because his pioneering work in the
isolation and characterization of bacterial diseases helped to identify the causes of many
of the maladies plaguing humanity. Further work by other scientists then began the long
road to conquering them.

1 - 11 Viruses are shown to parasitize organisms

 John Brown Buist was the first to observe a virus, the cowpox virus, although he
did not realize it.
 Dmitrii Ivanowski discovered that the cause of tobacco mosaic disease could
pass through a porcelain filter, but it was Martinus Beijerinck who correctly
deduced that the particle causing the disease was too small to be a bacteria and
would later be known as a virus.
 Frederick Twort and Felix d'Herelle each discovered bacteriophage, viruses that
infect bacteria.
The ensuing years brought numerous discoveries about the nature of disease. In the
latter half of the 19th century, the causative agents for anthrax, tuberculosis, gonorrhea,
diphtheria and many more were discovered. Great strides in understanding maladies
caused by bacteria were made at this time, including the solidification of the germ
theory of disease. However, some illnesses seemed not to have bacterial origins.
Microscopic examination of sera from ill patients revealed no organisms and the
causative agents could not be grown on any known medium. Yet if these sera were
injected into a susceptible host, disease resulted.

In 1886 John Brown Buist devised a method for staining and fixing liquid from a
cowpox vesicle. Observations of this slide showed tiny bodies that he believed were
spores. Although he did not realize it, he was the first person to see (and photograph) a
virus. It was not until the middle of the twentieth century with the invention of the
electron microscope that the true shape and structure of viruses was understood.

In 1884 Charles Chamberland in Pasteur's laboratory created an unglazed porcelain

filter that had pores much smaller than bacteria (0.1-1 µm). It was possible to pass a
solution containing bacteria through these filters and completely remove them from the
solution. This enabled the creation of sterile medium without the use of heat, and also
became a standard test for the removal of all bacteria, especially when testing
transmission of disease. By removing the microbes, Chamberland clearly demonstrated
that an infectious agent, and not the solution, was causing the illness. This concept
seemed reasonable until scientists began to investigate a tobacco infection.

Tobacco mosaic disease is characterized by light and dark green areas on plant leaves in
a mosaic pattern. The disease stunts the growth of the plant, therefore reducing yields.
Europeans recognized the effects of this disease soon after tobacco was introduced from
the New World in the 17th century. Adolph Mayer first described transmission of the
disease by injecting fluid from a diseased plant into a healthy one. Nine out of ten times
the healthy plant would become heavily diseased. In 1892 Dmitrii Ivanowski extended
this research by the shocking discovery that the causative agent could pass through
Chamberland's porcelain filter. He reported his findings, but the idea that the causative
agent could pass through the filter was so troublesome to Ivanowsky he attributed the
phenomenon to a cracked filter or to small spores that passed through the pores. It was
Martinus Beijerinck in 1898 who realized the true nature of these particles, making the
intellectual leap that the causative agent of the disease must be so small as to pass
through the filter known to trap all bacteria. He coined the term contagium vivum
fluidum a contagious living fluid.

Some scientists thought these agents were toxins in the fluids of the hosts, but further
study revealed that tobacco mosaic disease could cause illness through many transfers
from plant to plant. A toxin might cause damage on the first plant, but subsequent
transfers should make it so dilute that it would no longer have any effect. These
filterable entities were different from bacteria and appeared to depend on their host in
order to multiply. The term ultrafiltrable viruses and later just viruses (virus means
poison in Latin) was coined to describe these tiny pathogens.

Bacteria are also vulnerable to viruses. Bacterial viruses were described by Frederick
Twort in 1915 and independently by Felix d'Herelle in 1917. Felix d'Herelle was
studying a plague of locusts in Mexico when he noticed that the locusts were being
killed by a microorganism. During experiments to characterize this microbe, d'Herelle
noticed the appearance of clear, circular spots two or three millimeters in diameter on
cultures growing on agar. At this point d'Herelle studied the spots enough to determine
that they came from an agent small enough to pass through a porcelain filter. He
dropped the investigation, but recalled these observations while studying dysentery in
1915. The illness was affecting soldiers fighting in World War I and he soon determined
the cause to be the bacterium Shigella dysentery. In the process of investigating
Shigella, similar areas of clearing were observed. d'Herelle realized that something in
these areas of clearing was killing the bacterium. He eventually determined these were
viruses of bacteria, coining the term bacteriophage (devourer of bacteria). Figure 1-22
[28] lists these and other significant events in the discovery of the cause of disease.

Figure 1-22 Important events in the discovery of the cause of disease.

Year Event
Ignaz Semmelweis shows that hand washing between visiting mothers can
1840s
prevent childbirth fever.
Dr. John Snow studies a cholera outbreak in the Soho neighborhood of London
1854 and determines it was caused by contaminated water at the Broad Street pump.
His methods found the field of epidemiology.
1857 Louis Pasteur develops the germ theory.
Joseph Lister develops the use of phenic acid (phenol) to treat wounds and for
1867
antiseptic surgery.
Gerhard Henrik Armauer Hansen discovers the leprosy bacillus (Mycobacterium
leprae) and demonstrates that leprosy is a contagious disease and not inherited as
1873
was the popular belief. In many countries leprosy is still called Hansen's disease
in his honor.
Robert Koch and Cohn identify a bacterium, Bacillus anthracis as the cause of
1876
anthrax and publish their research.
1882 Koch isolates the Tuberculosis bacillus, Mycobacterium tuberculosis.
Koch puts forth his postulates, which are standards for proving that a
1884 microorganism is the cause of a disease. Application of Koch's postulates
continues to reveal the association of many diseases with pathogens.
1886 John Brown Buist is the first person to see a virus.
Dmitri Ivanowski publishes the first evidence of the filterability of a pathogenic
1892
agent, the virus of tobacco mosaic disease.
Martinus Beijerinck recognizes the unique nature of Ivanowski's discovery. He
1899
coins the term contagium vivum fluidum - a contagious living fluid.
Friederich Loeffler and Paul Frosch discover that foot and mouth disease is also
1899
caused by a filterable agent.
1915-
Frederick Twort and Felix d'Herelle discover bacterial viruses.
1917
In the fall of 1918, as World War I was ending, an influenza pandemic of
unprecedented virulence swept the globe, leaving some 40 million dead in its
1918
wake. A search for the responsible agent began in earnest that year, leading to
the first isolation of an influenza virus by 1930.
1957 D. Carleton Gajdusek proposes that a slow virus is responsible for the wasting
disease kuru. In subsequent years several diseases are shown to be caused by
 slow viruses (later renamed prions) including mad cow disease.

1 - 12 The power of vaccination is discovered

 Variolation, immunization against smallpox, was a common practice before

vaccination was common. This worked because the patient was exposed to a
weak strain of smallpox, which did not kill, yet provided immunity to the
disease.
 Edward Jenner discovered that cowpox could protect against smallpox, with a
much lower incidence of complications than variolation.
 Pasteur discovered a general method for immunizing people against disease
while working on chicken cholera. He coined the term vaccination to describe
the technique.

As soon as it was realized that microbes cause illness, the search was on for ways to kill
or to prevent them from causing disease. In this section and the next, we will look at
two series of events that illustrate the emergence of modern treatment of infectious
disease: the development of vaccines and the discovery of antimicrobial compounds.

Smallpox was a feared disease throughout human history and justifiably so. It was
highly contagious and almost everyone eventually became infected. Mortality rates
were as high as 25% in adults and closer to 40% in children. Those who did survive
often had scarring due to the blister-like pustules that form on the skin, but they
obtained life-long immunity to the disease.

As far back at the 11th century in India and China it was realized that liquid from the
pustules of a smallpox victim, when scratched on the skin of a healthy patient, would
most often cause mild disease. This intentional infection, termed variolation, would also
give life-long protection against the virus. Lady Mary Wortley Montgue, wife of
ambassador to the Ottoman Empire, introduced variolation to England in 1721 and it
became a popular practice throughout Europe. Washington even began variolating the
Continental Army in 1776.

Variolation had some deleterious side effects. Serious skin lesions inevitably resulted at
the site of inoculation, often accompanied by a generalized rash or even a full case of
smallpox. The fatality rate from variolation was 1 to 2 %. Today we would find this
level of fatality to be unacceptable, but at the time this risk still represented a significant
advance.

In 1796, Edward Jenner, an English country physician pictured in Figure 1-8 [29], went
in search of a more predictable and safer method of protection against the disease. He
noticed that milkmaids rarely contracted smallpox. Further investigation revealed they
often contracted cowpox from their charges. Jenner hypothesized that cowpox was
related to smallpox and contraction of the former would protect against the latter. In a
classic experiment (and one that would land you in jail today), Jenner inoculated a
young patient with cowpox and later challenged him with smallpox. The boy did not
become ill and Jenner was responsible for the creation of a safer method of protection
against smallpox. It is important to stress that the nature of these diseases and their
viruses would not be known for over 100 years. Jenner was ahead of his time.
Figure 1-8 Edward Jenner

Beginning around 1876 Pasteur's studies on chicken cholera led to the development of
vaccines to fight the disease. Cholera was a serious problem since it was able to spread
through a barnyard and wipe out a flock in as little as 3 days. It was transmitted by
contaminated food or animal excrement. Pasteur identified the cholera bacillus and grew
it in pure culture. When injected with it, a chicken invariably died within 48 hours.

Then, as often happens in scientific research, luck intervened. During the heat of the
summer, Pasteur returned to Paris and left the cholera cultures used for infection stored
on the shelves of his laboratory in Arbois, France. Upon returning, something had
happened to the cultures, they no longer caused disease when tested in chickens. With
some impatience for the time they were wasting, his group set to work making new
cultures of the bacillus and tested these batches on both new birds and also those
previously inoculated with the ineffective strain. To their amazement the previously
injected birds were unaffected by the fresh bacillus culture, while the new birds all died.
Pasteur immediately realized that this was similar to the studies of Jenner.

Pasteur then developed a method for creating cultures that would confer immunity, but
not cause disease. Sometimes this involved growing the microbe in medium in the
laboratory where they would spontaneously loose their virulence. In other cases it
involved multiple passes through a susceptible host. For example, the rabies virus was
attenuated by passing it through rabbits. In honor of Jenner's accomplishments Pasteur
coined the term vaccination (vacca = cow in Latin) for the process of immunization
against disease. In this and several of Pasteur's other discoveries, luck played a part, but
it was only helpful because he tenaciously pursued "odd" results and had the insight to
arrive at important conclusions. In Pasteur's famous words, " In the field of observation,
chance favors only the prepared mind."
Pasteur's technique of weakening a strain by a damaging treatment or passing it through
a susceptible host was termed attenuation and resulted in the creation of vaccines
against anthrax, plague, yellow fever, rabies and many other diseases. Many vaccines
have been developed over the years and children today receive a number of shots,
greatly decreasing infant mortality.

1 - 13 Antimicrobial compounds are developed to kill microorganisms

 Paul Ehrlich spent 17 years in search of a chemical treatment against syphilis,

eventually discovering salvarsan.
 Alexander Fleming discovered the first antibiotic, penicillin.

We now pick up another thread though the web of microbial history that began with the
work of Paul Ehrlich. By 1885, it was becoming clear that the causative agents of many
illnesses were microorganisms. As scientists manipulated these microbes in the lab they
found that certain dyes and other compounds were able to inhibit their growth. This
inspired Paul Ehrlich to propose that chemicals may exist that kill the microbe, but not
the patient, thus curing the illness.

One of the diseases Ehrlich hoped to cure was syphilis, which had reached epidemic
proportions in Europe. Little did he realize it would be a 17-year odyssey before he
would develop salvarsan, the first effective chemotherapeutic agent. Salvarsan was the
606th chemical he tried. This arsenic compound effectively kills Treponema pallidum,
the causative agent of syphilis.

The treatment, however, had many problems, causing long lasting health complications
for those individuals who used it. In addition, despite Treponema being quite sensitive
to salvarsan, the physician had to administer it intravenously for optimum effectiveness.
Intravenous injection was a recent development and many doctors were leery of trying
the procedure. In London, a young physician by the name of Alexander Fleming, then in
the Army Medical Corps, was one of the few that was willing to treat patients. Fleming
even got the nickname private 606 from his burgeoning practice. His work validated the
effectiveness of salvarsan against syphilis and convinced others to administer the
treatment.

Fleming, pictured in Figure 1-9 [30], was a physician by training, but spent most of his
time studying bacteria, and his success with salvarsan motivated him to search for other
antibacterial agents. His first discovery was lysozyme, an enzyme produced by many
organisms including humans, which lysed some bacteria. This enzyme is not useful as a
therapeutic agent because it is difficult to administer as a drug, but Fleming did develop
titration methods and assays that would become very useful.

Figure 1-9 Alexander Fleming

Fleming's arguably most important contribution to science is his discovery of penicillin.
In September of 1928, before leaving on a summer holiday, Fleming streaked some
plates of Staphylococcus aureus and left them to incubate until his return. In an
improbable set of circumstances, the beginning of the holiday was cold, allowing some
contaminating mold spores (that had blown in from a nearby window) to grow up on
some of the plates. The temperature then increased encouraging the growth of the
Staphylococcus. Many experimenters when confronted with a contaminated plate look
for the trash bin, but Fleming instead spent some time examining it. The fungus had a
zone of clearing around it where the Staphylococcus colonies would not grow,
suggesting the fungus was producing an antibacterial compound that had diffused into
the medium. Intrigued, he cultured the fungus, a Penicillium mold, and eventually
isolated a soluble extract that could kill bacteria and treat localized infection. He called
the new compound penicillin after the mold from which it came. Due to the technology
then available, however, it was very difficult to prepare a solution that could be used
throughout the body without causing problems.

World War II added a greater urgency to the search for compounds that could fight
infectious disease. Wounded soldiers, if they survived the initial injury, would often
develop life-threatening infections and there were no effective drugs to combat them. In
1939 Howard Florey and Ernst Chain began a systematic study of antimicrobial
compounds in hopes of developing treatments for these soldiers and ran across
Fleming's report written 9 years earlier. They now were able to purify the compound
completely and describe its high potency against microbes. The availability of penicillin
during World War II saved countless lives. The rediscovery of penicillin touched off a
search for other microbes producing substances that could kill or inhibit microbes,
leading to the discovery of many more antimicrobials. With Florey and Chain, Fleming
was awarded the Noble prize in Medicine and Physiology in 1945.

In the ensuing decades the search and discovery of numerous antimicrobial compounds,
combined with the development of vaccines, has eliminated many of the deadly diseases
that plagued humankind. While we now realize this is a continuing war and not a one-
time battle, our understanding of these microbes and the nature of disease will likely
keep infectious disease at bay for the foreseeable future. Figure 1-23 [31] lists important
events in the treatment and prevention of disease.

Figure 1-23 Treatment and prevention of disease

Year Event
Physicians in India and China realize that the liquid from the pustules of a
smallpox victim, when scratched on the skin of a healthy patient, would most often
1100
cause mild disease. This intentional infection, termed variolation, would also give
life-long protection against the illness.
Lady Mary Wortley Montgue, wife of the ambassador to the Ottoman Empire,
1721
introduces variolation to Europe.
1796 Edward Jenner uses cowpox to immunize against smallpox.
Ilya Ilich Metchnikoff demonstrates that certain body cells move to damaged areas
of the body where they consume bacteria and other foreign particles. He calls the
1884
process phagocytosis. This is the beginning of the science of immunology, the
study of the immune system.
Paul Ehrlich proposes that certain chemicals affect bacterial cells and begins a
1885
search for one that can treat syphilis.
Theobald Smith and D. E. Salmon develop a treatment for hog cholera by injecting
1886 killed hog cholera microorganisms into pigeons and demonstrate immunity to
subsequent administration of a live microbial culture of cholera.
1891 Ehrlich shows that antibodies are responsible for part of immunity.
Almwroth Wright and David Sample develop an effective vaccine against typhoid
1897
fever using killed cells of Salmonella typhi.
1897 Waldemar Haffkine develops a vaccine against the plague.
Paul Ehrlich announces the discovery of a cure for syphilis. The cure is the first
1912
specific chemotherapeutic agent for a bacterial disease.
1929 Alexander Fleming publishes the first paper describing penicillin.
Gerhard J. Domagk uses Prontosil, a chemically synthesized antimetabolite, to kill
1935
Streptococcus in mice.
Max Theiler produces a vaccine against yellow fever by passaging the virus
1938
through mice to weaken it.
Howard Florey and Ernest Chain produce an extract of penicillin and show it can
1940
kill bacteria in animals.
Ernest Chain and E.P. Abraham describe a substance from E. coli that can
1940 inactivate penicillin. This demonstrates how rapidly bacteria can become resistant
to antibiotics.
Selman Waksman and H. Boyd Woodruff discover actinomycin, the first antibiotic
obtained pure from a group of soil organisms, the actinomycetes. In subsequent
1940
years many antibiotics are isolated from this group including tetracycline and
streptomycin.
Charles Fletcher demonstrates that penicillin is non-toxic to human volunteers, by
1941
injecting a police officer suffering from a lethal infection.
1942 Selman Waksman suggests the word "antibiotic" to describe the class of
compounds produced by one microorganism that inhibit or kill other
 microorganisms.
Albert Schatz, E. Bugie, and Selman Waksman discover streptomycin, a very
1944
effective drug against tuberculosis.
W. H. Feldman and H. C. Hinshaw at the Mayo Clinic successfully treat
1944
tuberculosis with streptomycin.
The Soviet delegation to the World Health Organization proposes a vaccination
1957
effort to eradicate smallpox. The program finally begins in 1967.
Ali Maow Maalin, age 23 of Somalia, is the last known victim of naturally
1977
occurring smallpox.
Smallpox is declared to be eliminated. This is the only example of a microbial
disease that has been wiped from the face of the Earth. (However, the recent
1979
specter of bioterrorism and the smallpox stocks kept by several governments make
new epidemics of smallpox still possible.)

1 - 14 Beijerinck and Winogradsky initiate the field of environmental

microbiology

 Martinus Beijerinck and Sergei Winogradsky were two of the major scientists to
begin investigation of microbes in the environment that did not cause disease.
 Linus Pauling and Emile Zuckerkandl developed the idea of using the sequence
of biological macromolecules as a way of comparing and classifying microbes.
 Carl Woese and others then developed a classification system based upon 16S
rRNA. This system organized the tree of life and is used today to help classify
all organisms.

Contrary to what you would believe from news and advertising, the vast majority of
bacteria are harmless to humans and a large number are actually helpful.
Understandably, early work in microbiology focused on pathogenic microbes and
treatment of the diseases they caused. Martinus Beijerinck and Sergei Winogradsky
began the transition from this early work into the present era where we study the
molecular biology of a wide variety of microorganisms. Both were interested in
microbes present in the soil and water and their work founded the discipline of
environmental microbiology. Much of what we understand about bacteria in the
environment and their impact can be traced back to the efforts of these two scientists.

Martinus Beijerinck, depicted in Figure 1-10 [32], was originally trained as a botanist
and began his work studying the microorganisms that were present in and around plants.
He soon began experiments with microbes in the soil. His greatest contribution was the
development of enrichment media. Previously, microbes were cultivated on medium
consisting of potatoes or extracts of leftover animal renderings. Such media would
support the growth of many different bacteria, with chance and population density
dictating what became dominant in the culture. In many cases this was exactly what was
desired, but in other cases it was of interest to find bacteria capable of performing
certain chemical conversions.

Figure 1-10 Martinus Beijerinck

Beijerinck discovered that by adding or removing certain compounds from the medium
or incubating under different conditions, it was possible to favor the growth of certain
microbes and prevent the growth of others. An example is Beijerinck's work with
nitrogen-fixing bacteria, which are important in agriculture and the global cycling of
nitrogen. They are capable of taking nitrogen gas (N2) from the air and reducing it to
ammonia (NH3). This is an important property since most organisms, including
agriculturally important plants, can only use reduced nitrogen compounds such as
ammonia. Beijerinck wanted to isolate an organism capable of fixing nitrogen in the
presence of air, because all previous isolates fixed nitrogen only under anaerobic
conditions. By making medium that did not contain a source of fixed nitrogen and then
incubating in the presence of air (containing N2), he demanded that any microbe
growing in the medium had to be able to derive its nitrogen by performing aerobic
nitrogen fixation. Using this method he succeeded in isolating a new microbe
(Azotobacter chroococum) with these capabilities. Similar selective culture techniques
(as he liked to call them) enabled his laboratory to isolate sulfur-reducing and sulfur-
oxidizing bacteria, Lactobacillus species, green algae and many other microbes.
Selective culture techniques have since been applied to many different groups of
microorganisms, allowing them to easily be brought into pure culture.

Sergei Winogradsky, pictured in Figure 1-11 [33] was also interested in soil bacteria,
especially those involved in the cycling of nitrogen and sulfur compounds. He was one
of the first to isolate microorganisms responsible for the conversion of these elements in
the soil, obtaining pure cultures of bacteria capable of oxidizing ammonia to nitrate.
Winogradsky also studied the consumption of hydrogen sulfide gas by sulfur-oxidizing
bacteria directly in their natural habitat.

Figure 1-11 Sergei Winogradsky

When working with Beggiatoa (one of these sulfur-oxidizing bacteria) he discovered it
was obtaining its cell carbon from carbon dioxide. He used the term autotrophy to
describe this property, a radical idea at the time, and one not readily accepted by other
microbiologists. This led Winogradsky to propose the concept of chemolithotrophy;
bacteria capable of growth using purely inorganic sources of carbon and energy. He
realized that these microbes were essential for the cycling of nutrients in the
environment, and the living world was dependent upon their action, long before others
accepted the idea.

As a result of the work of Winogradsky and Beijerinck there was great enthusiasm for
identifying and classifying the bacteria inhabiting our natural world. For the first part of
the 20th century many scientists isolated microbes and made proposals for their
organization into genera and species. In 1909 Sigurd Orla-Jensen suggested a
classification scheme based on the functions and abilities of the bacteria, such as growth
on certain compounds or the production of specific by-products. The Society of
American Bacteriologists, later to become the American Society of Microbiology,
applied this technique to prepare a report on the classification of bacteria. This
eventually evolved into Bergey's Manual of Determinative Bacteriology in 1923 and
subsequent editions have become authoritative reference works.

There was great optimism that as more bacteria were brought into pure culture a clear
organization, based on the evolutionary history of microbes, would emerge from studies
of their physiology. However, by the 1940's it became clear that bacteria were unwilling
to go along with this idea because classification based on one set of properties was often
inconsistent with classification using a different set of properties. Scientists threw up
their hands and gave up the idea of finding a system of classification based on growth
and morphological characteristics.

Figure 1-12 Carl Woese

Several decades passed during which bacteria became tools for understanding life, but
their place in evolution was of little interest. Then in 1961 Brian McCarthy and E. T.
Bolton developed methods for the comparison of genetic material between species and
Linus Pauling and Emile Zuckerkandl formalized the idea of using the makeup of
biological molecules, their sequence, as a way to determine phylogenetic relationships.
Later, Carl Woese, pictured in Figure 1-12 [34], began to use this insight, choosing the
sequence of ribosomal RNA as the basis for evolutionary comparisons. His initial
efforts used the smaller RNA, termed 16S, of bacterial ribosomes, which he showed to
be a marvelous "evolutionary clock." This analysis was a watershed event for the
evolutionary classification of bacteria and in 1977 Woese used this technique to assert
that known bacteria should be divided into two separate domains that we now call
Bacteria and Archaea, radically changing our view of the microbial world. Because all
organisms have ribosomal RNA, this method of analysis has since been applied to the
identification and classification of large classes of organisms such as plants and birds,
fundamentally reshaping our views of the biological world. As you will read in Figure
1-24 [35] describes significant events in environmental microbiology.

Figure 1-24 Significant Events in Environmental Microbiology.

Year Event
1887 Sergei Winogradsky studies Beggiatoa and establishes the concept of autotrophy.
1888 Martinus Beijerinck develops the technique of enrichment culture.
Winogradsky discovers the organisms responsible for nitrification is soil, which is
1891
of great importance in agriculture because nitrogen is a limiting nutrient in the soil.
Martinus Beijerinck obtains the first pure culture of sulfur-oxidizing bacterium,
1904
Thiobacillus denitrificans.
Cornelius Johan Koning suggests that fungi are critical for the decomposition of
1904
organic matter.
Sigurd Orla-Jensen proposes the use of physiological characteristics for the
1909 classification of bacteria. He later publishes a monograph on lactic acid bacteria
that establishes the criteria for assignment.
The Society of American Bacteriologists presents a report on the characterization
1920 and classification of bacterial types that becomes the basis for Bergey's manual in
1923.
 Brian McCarthy and E. T. Bolton describe a method to compare genetic material
1961 from different species using hybridization. Using this technique it is possible to
quantitatively compare the relatedness of the two species.
Emile Zuckerkandl and Linus Pauling publish "Molecules as documents of
1965 evolutionary history", making a compelling case for the use of molecular
sequences of biological molecules to determine evolutionary relationships.
Don Brenner and colleagues establish a more reliable basis for the classification of
clinical isolates among members of the Enterobacteriaceae. They use nucleic acid
1969
reassociation, where DNA of one organism is allowed to hybridize with another
organism. This technique is used to help define a species.
Carl Woese uses ribosomal RNA analysis to identify a third form of life, the
1977 Archaea, whose genetic makeup is distinct from but related to both Bacteria and
Eucarya.
Holger Jannasch discovers abundant life at the bottom of the ocean near deep sea
1977 hydrothermal vents. The entire system is dependent upon sulfur oxidizing
microorganisms. Light and photosynthesis do not drive the process.
Karl Stetter isolates hydrothermophilic microbes (Archaea) that can grow at
1982
105°C. The discovery redefines the upper temperature at which life can exist.
Gary Olsen, Carl Woese and Ross Overbeek summarize the state of phylogeny in
1994 prokaryotes. This causes scientists to rethink the classification of life and
emphasizes the importance of microbes.

1 - 15 Studying microbes provides insight into life at the molecular level

 Microbes have been critical tools for the understanding of basic biology at the
molecular level.
 Fred Griffith performed transformation experiments that suggested that DNA
was the hereditary material. His student, Avery Oswald, later demonstrated this
conclusively.
 Beadle and Tatum developed the one gene-one enzyme hypothesis using
Neuropsora crassa as the test microbe.
 Many scientists working with E. coli deciphered the nature of DNA, RNA and
protein. These experiments lead to our understanding of how information in the
cell is stored and how it is converted into proteins.

In the early part of the 20th century techniques were developed to examine the inner
workings of the cell and much of the work was performed in bacteria due to their
experimental accessibility. Before this period, the method of turning genetic information
into the proteins that carry out cellular processes was completely unknown. Indeed, the
chemical composition of the genetic material was being hotly debated. From Gregor
Mendel's work with pea plants, the nature of inheritance and heredity was understood.
However, it was unclear what molecule in the cell maintained and passed hereditary
information on to subsequent generations. The leading contender for this role was
protein, when in 1928 Fred Griffith discovered transformation in bacteria.

Griffith knew that when Streptococcus pneumoniae strains are injected into mice, they
cause rapid deterioration and death. Griffith isolated strains of the bacteria that were no
longer capable of producing an outer slime layer and appeared rough when grown on
solid medium in contrast to the smooth colonies of the original isolate. When these
rough strains were injected into mice, no illness resulted. Similarly, when a smooth
microbe was heat-killed and then injected, the mice showed no signs of infection. A
surprise was waiting for Griffith when he injected into mice a mixture of dead cells of a
smooth isolate with live cells of a rough isolate: they died. When bacteria were isolated
from these dead mice, they formed smooth colonies. Griffith hypothesized that the
ability to create the slime layer was passed from the dead smooth cells to the viable
rough cells, making them pathogenic again. The process became known as
transformation.

Griffith was ridiculed, as most scientists believed his preparation were contaminated
with viable smooth cells. It was not until 1944 that his student Oswald Avery and
coworkers repeated the experiments of Griffith, reproducing his results and discovering
that DNA was the material from the dead smooth cells that transformed the rough
mutant. This was strong proof that the hereditary material in cells was DNA. Further
experiments in the 1940's clearly established this observation with Joshua Lederberg
discovering two more ways that DNA could be transferred between bacterial cells,
conjugation and transduction. For more information on these experiments, see the
chapter on Genomics and Genetics

In 1943 Beadle and Tatum reported experiments with the fungus Neuropsora crassa
that eventually established the idea that each gene in the DNA typically codes for one
protein (the one gene-one enzyme hypothesis). About 10 years later, Francis Crick,
Rosalind Franklin, James Watson and Maurice Wilkins worked on experiments
describing the structure of DNA and making predictions about how it was replicated. It
was now clear that DNA stored the information for proteins and that proteins performed
the many functions of the cell.

The important question now became, how does one convert the information in DNA
into protein. A major contribution in understanding this puzzle was made by Paul
Zemecnik and his laboratory who developed cell-free systems, first with rat liver and
later using the bacterium E. coli. This allowed the study of translation in the test tube
and he and other scientists used these systems to discover the important molecules
involved in the process. An intense effort to describe these molecular events then
ensued. The first insight came when Crick, Sydney Brenner and colleagues proposed the
existence of transfer RNA (tRNA), a molecule that helps to create amino acid polymers
based on nucleic acid sequence. Another critical player in the processing of information
was revealed when Brenner, Francois Jacob and Matthew Meselson discovered that
translation of genetic material into protein takes place on the ribosome and that the
molecule being translated at the ribosome is RNA, not DNA. The next mystery was
solved by Marshall Nirenberg and J. H. Matthaei when they developed methods to
decipher the genetic code that dictates the correspondence of nucleic acids to amino
acids. After the stunningly productive decade of the 1960's, the nature of the framework
for the conversion of the genetic information into proteins was now understood and the
basic mechanism has since been shown to be conserved across all biology. For more
information on the process of transcription and translation, see the chapter on the
Central Dogma

Figure 1-25 [36] lists important events in learning about the central molecular events in
biology. Note that almost all of this work occurred in microorganisms.
Figure 1-25 Significant Events in Learning about the Molecules of Life

Year Event
Frederick Griffith discovers transformation in bacteria and establishes the
1928
foundation of molecular genetics.
1941 George Beadle and Edward Tatum develop the one-gene one-enzyme hypothesis.
Salvador Luria and Max Delbruck demonstrate that inheritance in bacteria follows
1943
Darwinian principles.
Oswald Avery, Colin MacLeod, and Maclyn McCarty show that DNA is the
1944
hereditary material.
Joshua Lederberg and Edward L. Tatum discover a second method of gene transfer
1946
in bacteria: conjugation.
Joshua Lederberg and Norton Zinder find a third method of DNA transfer in
1952
bacteria using bacteriophage: transduction.
Alfred Hershey and Martha Chase suggest that only DNA is needed for viral
1952
replication.
1953 Francis Crick, Maurice Wilkins and James Watson describe the structure of DNA.
1954 Paul Zemecnik develops a cell-free system for translation using rat liver
Francis Crick, Sydney Brenner, and colleagues propose the existence of RNA that
1954
helps to transfer the information in DNA into protein (tRNA).
1956 Paul Zemecnik discovers tRNA in his cell-free system
1957 Francois Jacob and Elie Wollman show that the chromosome of E. coli is circular.
Peter Mitchell proposes the chemiosmotic theory in which a molecular process is
coupled to the transport of protons across a biological membrane. He argues that
1959
this principle explains ATP synthesis, solute accumulations or expulsions, and cell
movement.
Arthur Pardee, Francois Jacob, and Jacques Monod show that lactose induces β-D-
1959
galactosidase the catabolic enzyme that begins the degradation of the sugar.
Arthur Kornberg demonstrates DNA synthesis in cell-free bacterial extracts and
1960
later shows a specific enzyme complex catalyzes the synthesis of DNA.
Francois Jacob, David Perrin, Carmen Sanchez and Jacques Monod propose a
1960 mechanism for the for control of bacterial gene expression in an organization they
call the operon.
Paul Zemecnik and Robert Lamberg develop a bacterial cell-free system using E.
1960
coli
Marshall Nirenberg and J.H. Matthaei observe that a synthetic polynucleotide,
composed only of a string of the base uracil, directs the synthesis of a polypeptide
1961
composed only of phenylalanine. This begins the quest to unravel the genetic code
that translates DNA into protein.
Sydney Brenner, Francois Jacob and Matthew Meselson show that ribosomes are
1961 the site of protein synthesis and that RNA carries messages from the DNA to the
ribosome.
Lynn Margulis proposes that endosymbiosis has led to the generation of
1968
mitochondria and chloroplasts from bacterial progenitors.
Howard Temin and David Baltimore independently discover reverse transcriptase,
1970
a radically different way to alter genetic information in cells.
Thomas Cech and Sidney Altman independently show that RNA, and not just
1975
protein, can serve directly as a reaction catalyst.
1 - 16 Work on microbes paves the way to taking control of the genome

 E. coli continues to help scientists answer many basic questions about biology
and is a indispensable tool in genetics and genomics.
 Joshua Lederberg discovered plasmids, small, circular, self-replicating pieces of
DNA.
 The discovery of restriction enzymes allowed scientists to build chimeric DNA
molecules, and gave birth to the technique of cloning. It was now possible to
isolate any DNA molecule, mix it with a plasmid and created recombinant
molecules. These could be grown in E.coli, and other microbes, to make as
many copies as the scientist needed.
 The polymerase chain reaction allows the amplification of any DNA sequence
over a million fold. °CR has found hundreds of applications in science and
medicine.
 DNA sequencing, independently developed by Walter Gilbert and Fred Sanger,
has allowed the determination of the sequence of millions of base pairs of DNA.
Genomic sequences of organisms, including humans and many important
pathogens, are generating new insights into us, as well as the diseases caused by
certain microbes. This large amount of data has spawned the fields of genomics
and bioinformatics.

One final thread we will trace is the rise of molecular microbiology and the technique of
genomics. This pathway begins in 1885 with the seemingly unimportant isolation of a
common intestinal microorganism by Theodor Escherich. The microbe is eventually
renamed Escherichia coli in his honor. It could not have been understood at the time,
but this bacterium would play a central role in our understanding of life. E. coli
achieved this position because it has several properties that made it invaluable as an
experimental system:

1. E. coli is easy to take care of. It can grow on a wide variety of media, at
temperatures ranging from 20-45 °C, in the presence or absence of air.
2. E. coli grows quickly. Under normal laboratory conditions it goes through a
complete life cycle and double its population about every 30 minutes. With this
rapid growth rate it is possible to generate large populations in just a few hours.
Most experiments can be set up one day and the results observed the next.
3. It can survive storage for long periods of time without losing viability using
relatively simple preservation techniques.
4. It was available. There is an element of arbitrariness that this microbe came to
serve such a vital role in biology. Many other bacteria probably could have filled
this position, but E. coli was the one chosen.

Several key discoveries laid the foundation for future developments in molecular
biology. The first of these was the discovery of autonomously replicated pieces of DNA
separate from the bacterial chromosome. plasmids, as Joshua Lederberg called them,
were capable of carrying genes that could be regulated and moved from one microbe to
another independently of the bacterial chromosome. The ability of some plasmids to
move between different cells meant that different bacteria could rapidly inherit genes
carried on these plasmids. It was found that one class of plasmids encoded resistance
genes that would enable the bacteria to grow in the presence of an antibiotic. It later
became clear it was possible to select for the presence of these plasmids by demanding
growth of the bacterial strain on medium containing the antibiotic to which the plasmid
encoded resistance.

A second discovery that greatly impacted modern genetic engineering was that bacteria
have their own parasites. As mentioned earlier, there are viruses that infect and kill
bacteria, just as viruses infect animals and plants. The analysis of bacterial viruses was a
major avenue to understanding the bacteria themselves.

For example, investigation of the ability of some viruses to grow well on certain strains
of bacteria and not others lead to the discovery of restriction-modification systems in
bacteria. These systems allow bacteria to recognize and destroy foreign DNA and
consist of two parts. A modification enzyme that labels DNA as belonging to the
bacterium and a restriction enzyme that recognizes a certain 4-8 base pair sequence in
the DNA and cuts it in two if it has not been modified. The landmark paper of Hamilton
Smith and Kent Wilcox showed that restriction enzymes were capable of cleaving
double-stranded DNA into discrete pieces. The authors correctly hypothesized that the
enzyme was recognizing a sequence of DNA and cutting it.

Further studies on restriction enzymes led to the birth of genetic engineering. Janet
Mertz and Ronald Davis made restriction enzymes into tools for the manipulation of
DNA when they showed that many of the breaks these enzymes made in the DNA
produced single-stranded complementary ends. Stanley Cohen, Annie Chang, Robert
Helling and Herbert Boyer then demonstrated that any DNA can be broken into
fragments with restriction enzymes and by mixing that with a plasmid digested in a
similar manner, it was possible to create recombinant molecules. Moving the plasmid
into a microbe and growing it on selective medium could produce any desired amount
of these recombinant molecules.

Another discovery was "brewing" in the western United States. In the mid 1960s,
Thomas Brock was on a trip to Yellowstone and became intrigued by the mats of green,
brown and pink material that were found in many of the hot springs. Brock was sure
that these were living communities of microorganisms, yet the springs were at
temperatures near boiling. Subsequent research proved his hypothesis correct and lead
to the isolation of a microbe, Thermus aquaticus, capable of growth at 85 °C.

Organisms capable of growing at this high a temperature were unheard of at the time
and the discovery spawned two major developments. First, work with T. aquaticus and
other unusual microbes by Woese and many others lead to the discovery of the Archaea.
Second, the enzyme responsible for replicating DNA in T. aquaticus was found to be
extremely heat stable, surviving temperatures of 95 °C and copying DNA at 72 °C. This
enzyme allowed the following important breakthrough. In the early '70s, H. Gobind
Khorana and colleagues had suggested a method to amplify DNA in a test tube. As they
envisioned it, the method used short oligonucleotides, DNA polymerase and repeated
cycles of heating and cooling. However, the polymerase was killed during each heating
step, so massive amounts of prohibitively expensive enzyme were required. When Kary
Mullis reinvented the method in 1985, the thermostable polymerase of T. aquaticus was
readily available, which made the procedure wildly easier and cheaper to apply. He
termed it polymerase chain reaction (always referred to as °CR) and it has become a
pivotal technique in many areas of science, from detecting bacteria in the environment
to the analysis of evidence at crime scenes.
In 1977 Walter Gilbert and Fred Sanger independently developed methods for
determining the exact sequence of bases in DNA. These techniques became
immediately useful in determining the sequence of numerous important genes being
investigated in countless laboratories.

Further refinement has made DNA sequencing more efficient. It became efficient
enough that in 1985 Robert Sinsheimer convened a meeting of a number of biologists
active in genetics and gene mapping. Out of this meeting came a proposal to sequence
the entire three billion base pairs of the human genome. This was equivalent at the time
of proposing a mission to the moon, technically possible, but with unpredictable value.
The initial cost of the project was estimated to be $10 a base pair or a staggering 30
billion dollars. There was resistance from a significant portion of the scientific
community, fearing that the project would siphon funds away from other worthy
scientific projects. However, the clarity of the goal ignited the imagination of Congress
and the public, creating unstoppable momentum for the project. After about a decade of
effort, a preliminary draft of the entire genome was released in the spring of 2001,
earlier than projected and dramatically under budget. The project itself drove the
development of sequencing technology, allowing the determination of millions of base
pairs of sequence per day at a cost of less than 3 cents per base at large sequencing
facilities.

Along with the human genome, hundreds of other organisms have already been
sequenced and there will be thousands of sequenced genomes before the decade is out.
These include the genomes of many pathogenic bacteria, the mouse, the fruit fly and the
nematode Caenorhabditis elegans, the last being an experimental model for
development in eukaryotes. The analysis and application of this sequence data to
investigate biological problems has resulted in the development of the field of of
genomics and bioinformatics. In their infancy, these fields are already producing
astonishing findings that are accelerating the progress of fields such as evolutionary
biology, immunology, bacterial pathology, bacterial physiology and cancer treatment.
Figure 1-26 [37] lists important events in understanding the genome.

Figure 1-26 Events in understanding genomes

Year Event
Theodor Escherich isolates a microbe from the colon that is later given the name
1885 Escherichia coli in his honor. This microbe later becomes the workhorse of
molecular biology.
Edward Buchner helps launch the field of enzymology by developing a cell
1897
extract from yeast that is able to ferment sugar to alcohol.
Salvador Luria and Mary Human, and independently Jean Weigle, describe
sensitivity in bacteriophage imposed by the host on which it was grown. The
1952 viruses are restricted to grow well only on specific strains of bacteria. This later
leads to the study of bacterial systems of restriction and modification, and
eventually the discovery of restriction enzymes.
O. Sawada and others demonstrate that antibiotic resistance can be transferred
1959
between Shigella strains and Escherichia coli strains by plasmids.
1966 Jon Beckwith and Ethan Signer move the lac region of E. coli into another
microorganism to demonstrate genetic control. It is quickly realized that
 chromosomes could be redesigned and genes moved.
Waclaw Szybalski and William Summers develop the technique of DNA-RNA
hybridization (mixing nucleic acids together and allowing them to base pair) to
1967
investigate the bacteriophage T7. This technique finds wide use in many
experiments.
Thomas Brock identifies Thermus aquaticus, a bacterium that grows at 85 °C.
1967 Heat-stable DNA polymerase is later isolated and used in PCR. Investigation of
this organism also leads to the discovery of the domain Archaea.
Werner Arber shows that bacterial cells have enzymes capable of modifing DNA
by adding methyl groups at cytosines and adenosines. This methylation helps the
1967
cell identify its own DNA. Accompanying nucleases recognize these sites and
cut the DNA if it is not methylated.
Hamilton Smith and Kent W. Wilcox describe the action of restriction enzymes,
1970 discovered by Arber, by the purification of one of these enzymes from
Haemophilus influenzae.
Joan Mertz and Ronald W. Davis establish that the RI restriction enzyme from
1972 Escherichia coli cuts at a specific site on the DNA. They also reveal that the
cleaved ends of the DNA are complementary, opening the way for cloning.
Paul Berg creates the first recombinant DNA molecule from viral and bacterial
1972
DNA.
Stanley Cohen, Annie Chang, Robert Helling, and Herbert Boyer develop the
1973
process of gene cloning.
The Asilomar Conference is convened to discuss possible problems associated
1975-
with gene cloning. A one-year moratorium is suggested, as well as guidelines for
1976
cloning research and for genetic engineering.
Walter Gilbert and Fred Sanger independently develop methods for determining
1977
the sequence of DNA.
The U. S. Supreme Court rules that microorganisms altered in the laboratory can
1980
be patented.
U. S. Pharmaceutical manufacturer Eli Lilly markets the first genetically-
1982
engineered human insulin.
Jeff Schell and Marc Van Montagu, Mary-Dell Chilton and colleagues move
1983
genes into plants.
Kary Mullis uses a heat-stable enzyme from Thermus aquaticus to establish PCR
1988
technology.
The entire sequence of one of the sixteen chromosomes of the yeast
1992
Saccharomyces cerevisiae is determined.
Craig Venter, Hamilton Smith, Claire Fraser, and colleagues at TIGR elucidate
the first complete genome sequence of a microorganism, Haemophilus
1995 influenzae. In the ensuing years many laboratories have produced sequences for
dozens of microbes including many important pathogens and those of industrial
or environmental importance.
The human genome project begun in 1990 finished a working draft of the entire
2000
human genome.

Let's end this chapter with a defense of fundamental scientific research. That is, research
without an obvious applied goal or application. If you look at the initial results
generated in many of these important analyses, the research would have seemed esoteric
and inconsequential. Is the analysis of microbes in the colon important? Who cares why
a virus can grow in one strain of bacteria and not another? Why should the government
support Tom Brock's isolation of microbes from the hot springs of Yellowstone
National Park? What use is knowing the DNA sequence of a virus or a bacterial operon?
Yet, each of these insights began a thread of inquiry that changed the world. The take-
home message is that basic research creates insights and applications beyond the
imagination of even the scientists performing the work and should be supported.

1 - 17 Summary

Microbiology is the study of organisms that at some point in their life exist as single
cells and contain a nucleic acid genome that can replicate. Many organisms fall into this
definition including algae, fungi, protozoa, bacteria and archaea. Together these
organisms have a profound impact on the biosphere, making up the majority of life both
in number and total mass. Many illnesses are caused by infection with microbes and
understanding these infections has lead to cures and better treatments. The emergence of
new infectious agents will spur continued interest in microbiology. Many more
microbes grow harmlessly in the environment, taking advantage of chemicals and/or
sunlight to grow. Research into these microbes has also helped us understand the basic
framework of life and revealed the basic fundamental rules that govern living systems.
In the past microbes have been used in experiments to answer many scientific questions
and they will continue to serve as excellent tools of inquiry in the future. A significant
number of these discoveries have lead to important applications in many areas of human
endeavor.

Chapter 2 - Cell Structure and Organization

2 - 1 Introduction

Microorganisms typically face the world as single cells rather than as the multi-cellular
assemblies of higher organisms. Each single cell [1] must therefore contain all the
structures necessary for managing its internal state and dealing with the outside
environment. Not surprisingly, this evolutionary process results in the use of rather
similar structures and processes to solve similar need in different microorganisms.
However, prokaryotes have been on this Earth for a long period of time and this has
allowed them to differentiate into a dizzying number of different species [2]. Eukaryotic
[3] microbes are not quite so diverse, but they still display a remarkable range of
properties. The diversity of the microbial organisms also means that this survey of
structures is not exhaustive. No one cell contains all the structures that we describe here,
but we will explore the more common structures that have been observed by scientists
in the past 150 years as show in Figure 2-1 [4]. A distinction in this discussion must be
made between the two types of prokaryotes: the Archaea [5] and their cousins, the
Bacteria [6]. We will initially focus on the Bacteria, since that is what we know the
most about. Many of the structures we will examine are found in both the Bacteria and
the Archaea, but there are some significant differences and these will be covered at the
end of the chapter. Finally, we will talk about the features that are distinctive among the
microbial eukaryotes.

Figure 2-1 The generalized bacterium

This cartoon displays many of the common structure found in prokaryotic
microorganisms, though not every one will have every one of these structures.

So how did scientists find out so much about such very small organisms? As you might
guess, many techniques come into play when tackling a subject as complex as bacterial
structure. Electron microscopes [7] have been important, of course, but so have
genetics, molecular biology and biochemistry. Microscopes help scientists to visualize
where these structures are located and how they are arranged spatially in the microbe.
Bacterial genetics and molecular biology identify and analyze the genes necessary for
the synthesis and regulation of these structures. Biochemistry permits the detailed
examination of each part separately, with implications for its role in the living
bacterium. The powerful combination of these disciplines has provided a deep
understanding of how a bacterium is put together, but there is still much to learn.

This chapter on microbial structure is separated into two sections [8]. In the first section,
we describe the chemical nature of the types of molecules and polymers that are
important in carrying out the business of biology. In the second section, we examine the
functional units in the cell, describing how the various chemical structures in the cell
interact to carry out important cellular functions. In this discussion we assume that the
student has had an introductory chemistry course (at least in high school) and is
somewhat familiar with chemical notation.

First and foremost, it is important to point out that there are some universal structures
that all living cells contain. They are the basic building blocks of life: DNA [9], RNA,
protein [10] and cellular membranes. Most, but not all, bacteria also possess cell walls.
Beyond these essentials, the frequency of the rest of the structures we mention here
ranges in the bacterial world from quite common to very rare.

2 - 2 Sugars are common in the cell

 Sugars store carbon and energy and are part of cellular structures.
 Sugars in biological systems are 3 to 7 carbons and contain a carbonyl group.
 Sugars can be monomers or polymers of 2, 3 or more sugars.

Sugars serve three basic purposes in the cell: as carbon and energy sources, as reservoirs
of carbon and energy, and as parts of cellular structures. Large amounts of energy can
be extracted from sugars by processes referred to as catabolism [11], as discussed in
Chapter 9. This may explain why many microorganisms show a preference for sugars if
given a choice of energy sources. The term carbohydrate is often used to refer to them
because their chemical formula can be broken down into [C(H2O)]x where x is any
number greater than three.

Sugar Monomers

Single sugar molecules are typically 3 to 7 carbons long and are termed Figure 2-2 [12]
shows that each carbon on the sugar molecule is decorated with a hydroxyl group (OH)
except for one carbon that forms a carbonyl group. If the carbonyl group is at the end of
the molecule, it forms an aldehyde [13]; if the carbonyl group is in the middle of the
molecule, it forms a ketone [14] as indicated in Figure 2-2 [15]. All sugars can exist as
linear molecules in solution and those greater than 5 carbons long can also circularize
with the carbonyl group attacking a hydroxyl on one of the other carbons. The circular
sugar contains 5 to 7 members in the ring with one of the members being oxygen.
Glucose, fructose and ribose are some of the more common sugars found in the cell.

Figure 2-2 Common monosaccharides

The structures of some important monosaccharides are shown. These often serve as
building blocks for major structures in the cell, such as the cell wall and the genome.
The top row shows the linear forms and the bottom row shows their circular forms.

Sugar Polymers

Sugars readily polymerize: the combination of two sugars is called a disaccharide, while
the combination of three is called a tri saccharide, and polymers of greater than three
sugars are referred to as polysaccharides. Sugars are connected by α or β linkages. If the
hydrogen is pointing up, it is a α linkage, if the hydrogen is pointing down, it is a β
linkage as shown in Figure 2-3 [16]. This distinction may seem trivial, but it greatly
influences the properties of the molecule. (The name of the linkage further depends
upon the orientation of the hydrogen on the lowest numbered carbon that forms the
bond. The lowest numbered carbon is decided by the rules of organic chemistry, but this
is really not important for our discussion. Just realize there is a set pattern of numbering
for each organic molecule and this helps determines the α or β linkage.) For example,
starch is made up of glucose linked by α-1,4 bonds. Starch is also water-soluble and can
serve as a food source for many organisms. In contrast, cellulose, containing glucose
linked by β-1,4 bonds, is insoluble in water and is not as readily degraded by most
microbes. Polysaccharides might contain only one type of sugar monomer or a variety
of different ones, sometimes in repeating units.

Figure 2-3 Common disaccharides, trisaccharides and polysaccharides

Sugars can be linked together to form more complex polymers. Lactose is a common
sugar in milk, while maltose is found in many grains. Starch found in potatoes and other
vegetables is a long polymer of glucose units. A common form of starch contains 20%
amylose and 80% amylopectin.

Polymers of sugars can serve as storage products for the cell. The breakdown of sugar
yields a huge amount of energy, which means that they are terrific molecules for
effectively storing energy for later utilization. Starch and glycogen are two examples of
sugar polymers that are used in this way. Polysaccharides also serve as structural
components of many different molecules in the cell including nucleic acids and the cell
wall.

2 - 3 Nucleic acids store information and process it

  DNA and RNA are composed of two parts, the sugar phosphate backbone and
nucleotide bases.
 The bases in DNA are purines (adenine and guanine) and pyrimidines (cytosine
and thymine). RNA contains the same bases, except uracil is substituted for
thymine and they contain an extra 5'-hydroxyl.
 DNA polymers can be thousands of base pairs long. RNA polymers are
hundreds to thousands of bases long.
 RNA is single-stranded. DNA is double-stranded and forms a double helix in the
cell.
 RNA molecules have significant secondary and tertiary structure that is
important in their functions.

Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are involved in information
storage and processing. DNA [17] serves as the cell [18]'s hereditary information, while
RNA is involved in converting that information into functional products, such as
proteins. RNA and DNA are long polymers of only 4 nucleotides [19]: adenine,
guanine, cytosine and thymine [20] (or uracil for RNA). Figure 2-4 [21] lists the
structure of the five nucleotides found in nucleic acids.

Figure 2-4 The structure of nucleotides.

The nucleotides for adenosine (A), guanine (G), cytosine (C ) and thymine (T) as found
in DNA are shown. The first three are also found in RNA, but when incorporated into
that polymer, the associated sugar has two hydroxyls, as shown in the model of uracil
(U). Uracil is the RNA equivalent of the DNA nucleotide thymine.

The nucleotide [22] structure can be broken down into two parts: the sugar-phosphate
backbone and the base [23]. All nucleotides share the sugar-phosphate backbone, while
the base distinguishes each type of nucleotide. Linking these monomer units together
using a 5’-oxygen on the phosphate and the 3’-hydroxyl group on the sugar forms the
nucleotide polymer as shown in Figure 2-5 [24]. Many thousands of DNA nucleotides
are strung together to form genes and chromosomes.
Figure 2-5 A schematic of the nucleic acid polymer

In this picture the bases of the two anti-parallel (running in opposite direction) strands
of a DNA double helix are shown. Note the sugar-phosphate backbone from which the
bases extend and pair with matching bases from the other strand. The dashed lines show
hydrogen bonds between the base pairs.

The bases of the four nucleotides are different, but there is also a pattern. Adenine (A)
and guanine (G) are purines [25], and therefore have a distinctive two-ring structure;
they differ in the chemical groups attached to the rings. Likewise, cytosine (C), thymine
(T) and uracil (U) are all pyrimidines [26] and share a single-ringed structure, but also
differ in their attached groups. Not surprisingly, as these extra chemical groups
distinguish the different purines and pyrimidines structurally, they are also responsible
for their important functional differences as well.

The bases in a nucleic acid polymer are capable of forming hydrogen bonds with
neighboring bases on a second strand of nucleic acid, a process termed base pairing
[27]. However, there are rules to this association. Adenine is capable of forming two
hydrogen bonds with thymine (or uracil) and cytosine can base pair with guanine,
forming three hydrogen bonds. Some of these bonds require the extra chemical groups
mentioned above. If two single strands of nucleic acid have sequences that can base pair
along the polymer (such sequences are sometimes said to complement [28]), they will
generate a long double-stranded polymer that has a staircase topology as shown in
Figure 2-6 [29]. This structure is termed a double helix, since two strands form the
molecule and it spirals around an axis in a regular pattern. Such a reaction between two
complementary [30] DNA strands is spontaneous: if you mix two complementary single
strands of nucleic acid together in a test tube at a reasonable temperature, pH [31] and
salt concentration, they will find each other and anneal to form a double-stranded
polymer.

Figure 2-6 The Double Helix

Note how the two nucleic acid strands spiral around each other in a regular repeating
pattern. There are 10 bases per turn of the double helix. Bases pair with one another in
the center of the helix cylinder and form what some have likened to a spiral staircase.
The above figure can be rotated by clicking on the arrows.

Secondary and Tertiary DNA Structures

DNA almost always exists in cells as a double-stranded structure of complementing

strands. It happens that this double-stranded form is rather stable [32], and resists tight
twists and turns. It is often assumed that the stability is due to the hydrogen bonding
between the bases, but this is not the case (the bases would hydrogen bond just as well
to water). Instead, it is largely due to the interaction between adjacent base pairs along
the helix, which is termed "base stacking [33]" Figure 2-7 [34] for an example. Finally,
the larger organization of the DNA strands with respect to each other, termed the
tertiary structure [35], is also fairly similar in all DNA molecules. One implication is
that proteins that want to distinguish between different DNA molecules must do so by
reading different primary structure [36] sequences by interacting with the outer surface
of the base pairs.

Figure 2-7 A molecular model of the lac repressor

The repressor binding to its recognition sequence. Any DNA-binding protein that
recognize specific DNA sequences must make specific contacts with that recognition
sequence by reading the pattern of bases in the DNA. These proteins do this by making
very specific contacts between themselves and those atoms on DNA that are different in
the different bases. In this figure, the DNA helix is shown by the ball-and-stick figure
running along the bottom and the protein is depicted by cylinders (which refer to alpha
helices) and other "squiggles" (which refer to any other structure in the protein). The
point of the figure are that the DNA is not opened up and that the recognition regions of
the proteins are very often alpha helices that lay parallel with or perpendicular to the
DNA helix. Not shown are the side chains of the amino acids that actual make the
specific contacts with the DNA. In this and subsequent molecular figures, of three
different displays will be shown: ball and stick, which shows all atoms larger than
hydrogen; ribbon models, in which a ribbon that runs along the carbon backbone of the
protein is displayed; and space-filling models, where the actual atomic surface of the
molecule is shown. Sometimes, as in this figure, the ribbon form is modified slightly be
showing cylinders for alpha helices instead of an actual helix as in Fig. 2-11. Most of
the structures shown in this text are the product of X-ray crystallography, which
essentially displays the position of every atom (larger than hydrogen) in a molecule.
Increasingly all biological questions are being thought about in terms of these structures
at the atomic scale.

Structures of RNA

In composition and therefore primary structure, RNA is similar to DNA, except that
uracil (U) takes the place of thymine in the molecule and the ribose unit on each sugar
contains a additional hydroxyl group. However, most RNA in cells exists as single-
stranded molecules and not a complex of two different strands as with DNA. Now if
complementary base sequences are present in an RNA molecule, it can fold back upon
itself and base pair, so that many RNA molecules have at least some double-stranded
regions. However, this bending and folding means that RNA molecules typically have
much more complicated tertiary structures than does DNA. Both the single-stranded
loops and the double-stranded stems are critical for the function of most RNA
molecules. Many are involved in creating physical structures, such as ribosomes, that
are involved in processing information. The other general class of RNAs are messenger
RNAs, which represent a version of the DNA primary structure that is suitable for
translation into protein.

2 - 4 Proteins are made of amino acids

 Amino acids are organic acids with a amine group attached to the α carbon.
 Amino acids are distinguished by their side group that dictates its properties.

Proteins and peptides (small proteins) are essential to the cell and serve two major
functions. Many proteins are enzymes that catalyze almost all biological reactions in a
living organism. Other proteins perform a structural role for the cell - either in the cell
wall, the cell membrane or in the cytoplasm [37]. In this section, we will look at the
basic structural elements shared by all proteins.

Structure of Amino Acids

Proteins are polymers of amino acids.. Amino acids, with rare exception, contain an α
carbon that is connected to an amino (NH3) group, a carboxyl group (COOH), and a
variable side group (R). Figure 2-8 [38] shows this generic amino acid structure. The
side group gives each amino acid its distinctive properties and helps to dictate the
folding of the protein.

Figure 2-8 A generalized amino acid

Each amino acid contains a carboxyl group, an amino group and a variable side group
(R). These all connect to a central carbon, termed the α-carbon, identified by the arrow.

2 - 5 The primary structure of proteins is the amino acid sequence

 Proteins contain 20 different amino acids that are linked by peptide bonds to
form long polymers.
 Amino acids can be classified into three groups, polar, non-polar, and charged.

As with nucleic acids, primary structure refers to the ordered sequence of the different
amino acids in a protein. The carboxyl group and the amino group of amino acids are
reactive. As shown in Figure 2-9 [39], cells synthesize proteins by attaching the
carboxyl group of one amino acid to the amine group the next, with polymerization
taking place at the ribosome. This is termed a peptide bond. Since each amino acid has a
carboxyl group and an amino group, hundreds of amino acids can be linked together.

Figure 2-9 A peptide bond

The peptide bond between an alanine residue and a valine residue is identified by the
arrow. Peptide bonds can form between any two of the 20 amino acids and link the
carboxyl group of one amino acid and the amine group of the next.

There are 20 common amino acids found in proteins and these amino acids can be
roughly classified into 3 groups: polar, non-polar and charged. Polar and charged amino
acids are hydrophilic and are often found on the surface of a protein, interacting with the
surrounding water. In contrast, non-polar (or hydrophobic) amino acids avoid water.
While this categorization is adequate for most purposes, you should recognize that it is a
bit simplistic. For example, arginine does have a charged hydrophilic group at one end,
but the -CH2- backbone that makes up most of the amino acid is actually quite
hydrophobic. Figure 2-10 [40] shows the chemical structure of all 20 common amino
acids.
Figure 2-10 The common amino acids

In the figure, the amino acids are organized into their chemical type and characteristics.
Acidic and basic amino acids carry a charge, amino acids with a sulfur or hydroxyl
comonent are polar, and aliphatic and aromatic amino acids are non-polar.

2 - 6 Secondary structure is the local geometry of the protein

 The chain of amino acids dictates how a proteins folds.

 The major determinant of protein folding is hydrophobic interactions.
 The local geometry of a protein is its secondary structure.
 Two common secondary structures are the α helix and the β sheet.

Peptides and proteins are formed when a ribosome and the rest of the translation
machinery link amino acids together in polymers that range from 10 to 10,000 residues
in length. During and after protein synthesis, the residues of the primary sequence
dictate how the protein folds. The simplest aspect of protein folding is termed its
secondary structure, which refers to the geometry of the local polypeptide chain with
respect to their immediate neighbors. How a protein folds is dictated by the primary
sequence of amino acids, but predicting the overall structure from the primary sequence
remains one of the most important unsolved problems in biology. Nevertheless, it is
clear that the major determinants of this final structure are hydrophobic interactions.
During protein folding, hydrophobic amino acids must be hidden from the water
interface by being buried in the interior of the protein. This burying defines the protein
core which then influences the immediate structure around it and greatly affects the
protein's overall structure.

Common Secondary Structures

Peptide bonds between adjacent amino acids can rotate and twist to allow a large
number of interactions, but two local organization schemes, the α helix and the β sheet,
are found in many proteins. Their prevalence is certainly because they happen to form
particularly energetically favorable structures. Formation of these structures is driven by
favorable hydrogen bonding and hydrophobic interactions between nearby amino acids
in the protein. The α helix resembles a ribbon of adjacent amino acids wrapped around a
tube to form a staircase-like structure. Figure 2-11 [41] shows different representations
of an α helix. This structure is very stable, yet cn be flexible in specific cases and is
sometimes seen in parts of a protein that may need to bend or move.

Figure 2-11 The α helix motif in proteins

This figure shows two common depictions of an α-helix, an extremely common and
important motif in proteins. The ball-and-stick depiction on the left shows the various
side chains, but the helical nature of the structure is not so obvious. The ribbon
representation on the right only traces the backbone of the peptide bonds and
overemphasizes the symmetry a bit, but shows the helical nature of the structure.

In the β sheet, two protein chains (perhaps different segments of the same protein) align
themselves in a planar structure such that hydrogen bonds can form between facing
amino acids in each sheet. Figure 2-12 [42] shows different representations of this
structure. The β sheet is different from the α helix in that it can involve amino acids
from different sections of the protein, which come together to form this structure. Also,
the structure tends to be rigid and less flexible than the α helix.

Figure 2-12 The β-sheet

Two views of a β-sheet. (a) A diagram of β sheet showing hydrogen bonding between
protein strands (b) A ribbon representation of a β-sheet.

2 - 7 Tertiary structure is the 3D structure of individual polypeptides

 Tertiary structure is stabilized by hydrophobic interactions, hydrogen bonds,

ionic interactions, and sulfhydryl bonds.
 Conserved tertiary structures are called motifs. There are many common motifs
that have been discovered in many proteins. Some examples are ATP-binding
and DNA-binding motifs.
 Structurally similar motifs imply a evolutionary relationship.
 Proteins are flexible, dynamic structures that respond to their surroundings.

The relationships mentioned above do not fully define the structure of proteins. For
example, we need a way to describe larger organizations of α helices and β sheets, as
well as other parts of proteins that do not fit these two patterns. This larger organization
is termed tertiary structure.

As noted before, the most important stabilizing force in proteins is burying hydrophobic
residues from the surrounding water, but there are other chemical features that are
important in creating and stabilizing tertiary structure. These include hydrogen bonds,
ionic interactions, and sulfhydryl bonds. Ionic interactions are attractions between
groups of opposite charge. There are amino acids with negatively charged side groups
(aspartate, glutamate) and amino acids with positively charged side groups (typically
lysine and arginine). If opposite charges are brought close enough together, there will be
an attractive force that can contribute to protein structure. It also follows that like
charges repel each other and this can also dictate protein structure. Sulfhydryl linkages
are covalent bonds between cysteine groups. Cysteine is a unique amino acid in that it
has a sulfur group at the end of its variable side group that is available for binding to
other groups. Often in proteins, nearby sulfhydryl groups on cysteines form a covalent
bond and these are often crucial for the stabilization of the mature protein or for it to
perform its function. Figure 2-13 [43] shows some representations of sulfhydryl bond in
proteins.

Figure 2-13 The chemical structure of a sulfhydryl bond

Several views of sulfhydryl linkages are shown. In the top left panel, the covalent
sulfhydryl linkage between two cysteine residues is shown. In the top right panel, this
sort of Cys-Cys bond is shown in the context of a section of a protein. The bottom panel
shows a ribbon diagram of a similar protein loop, but where the interacting sulfhydryl
groups on the side chains are shown in yellow.

Figure 2-14 [44] shows the tertiary structure of ribulose bisphosphate carboxylase
(RubisCo), one of the most abundant enzymes on this planet. This enzyme takes energy,
obtained most often from the sun, and uses it to convert carbon dioxide into
carbohydrate. Its abundance reflects the fact that virtually all photosynthetic organisms,
from bacteria to plants, use this enzyme to produce much of the cell's carbon. Note the α
helices throughout the protein and the β sheet near the bottom. These structures help to
hold the protein in its proper conformation so that it can carry out its enzymatic activity.

Figure 2-14 Tertiary structure of ribulose bisphosphate carboxylase

The figure shows the structure of ribulose bisphosphate carboxylase, typically
abbreviated Rubisco, from the bacterium Rhodospirillum rubrum. As described in the
text, this complex structure has alpha helices (shown as cylinders) and beta sheets
(shown as the flat ribbons) throughout.

Now we have talked about certain secondary structures that recur in many proteins, but
it also happens that there are conserved tertiary structures as well. In other words, there
are regions of proteins, which are sometimes termed motifs or domains, that are
structurally identical in two or more different proteins. Figure 2-15 [45] shows some
examples of protein motifs. By "structurally identical," we mean that they have
similarly sized α helices and β sheets that are organized in a similar overall way.
Interestingly, these domains typically do not have identical primary structures. Instead,
it appears that a variety of different sequences can fold to give the same overall tertiary
form. It is usually clear from the sequence that structurally similar domains from
different proteins are evolutionarily related. That is, at one time there was a single
protein with the domain and through evolution, an extra copy of the gene was created.
Subsequent random mutations in each gene altered the primary sequences of the
proteins, but the only changes that were tolerated were those that maintained the
structure, and therefore the function, of each copy of the domain. This implies that these
motifs often have a specific function. For example, there is a very common motif that
proteins use for binding ATP. A second class of examples would be the set of motifs
that are used to bind DNA. Through genetic changes, these motifs have been grafted
onto other protein regions, so that the same ATP-binding (or the DNA-binding) domain
performs a similar function for proteins with completely different overall activities. Of
course the unique functions are defined by those portions of the protein that are not in
common.
Figure 2-15 Some common protein motifs

Three examples of protein motifs are shown. The top left panel shows two zinc-finger
motifs (the red helices and thin gray lines), each bound to a Zinc atom shown in pale
blue. This is a common motif found in DNA-binding proteins and the interaction is
shown here (the ball-and-stick figure running from left to right in this panel is a section
of DNA). Note that the Zinc does not interact with the DNA, but instead stabilizes the
protein motif to allow it to form the precise shape to bind to DNA. The top right panel
shows an ATP-binding domain, with the ATP displayed as the space-filling model and
the protein shown in a ribbon format. The bottom panel shows another common DNA-
binding motif, termed the helix-turn-helix, as found in the lac repressor. Here the DNA
is shown in space-filling form and the two helices after which the structure is named are
shown in red and white (the latter is shown end-on and is interacting with the DNA).

The existence of these conserved motifs was revealed by the analysis of the crystal
structures of many different proteins. One saw similar structural regions performing
similar functions in proteins that otherwise were quite dissimilar. Not surprisingly, since
there are some residues that need to be conserved to form these structures, we can now
predict some of these motifs based on the sequence of the proteins as well. The
explosion of DNA sequence information and tools that can find these motifs has
allowed the prediction of protein functions solely from sequences to be done with ever
greater accuracy.

From this description you can see that proteins can often be looked at as a collection of
"protein pieces," each of which serves a function and has an evolutionary history. We
refer to these as functional domains. The enzyme pyruvate kinase, which generates ATP
by removing it from phosphoenol pyruvate, has three domains, termed A, B and C. The
A domain is responsible for binding substrate (phosphoenolpyruvate and ADP) while
the C domain serves a regulatory role, binding fructose-6-bisphosphate and stimulating
activity.

Up to this point, we have talked as if proteins are static rigid structures, but this is
certainly wrong. Instead, most proteins are flexible, dynamic structures that respond to
the conditions around them. Movement is typically essential to their function, especially
for enzymes that need to grab a substrate, react with it and release product. In some
cases, the flexibility is over very small distances; moving the residues of an active site
just a few tenths of an -10m')">angstrom [46] (10-10 meters) would have a major effect
on binding and reacting with a substrate that is itself only a few angstroms in size.
However, some protein movements are huge, where one domain changes its position
relative to that of the rest of the protein. This obviously changes the tertiary structure of
that protein. Thus, when we talk about tertiary structures, typically determined by
crystallography, we are really referring to only one of the possible structures that the
protein can exist in while performing its biological function. The structure we see
through crystallography is simply the form of the protein that happened to form crystals
and whose structure was then solved.

This flexibility has many important implications for a protein. At too high a temperature
the protein might become too dynamic. This might overwhelm the stabilizing energy
that maintains the protein, so that it unfolds or denatures. At too low a temperature, a
protein might be so stable that it locks into a single structure and lacks the flexibility
necessary to carry out its function. Interestingly, thermophilic microbes have been
found to have more rigid proteins that can tolerate the turbulent environment of higher
temperatures.

2 - 8 Quaternary structure is the total complex of a functional protein

 Proteins can contain several polypeptides that form a functional unit. These
protein complexes can be composed of several copies of the same polypeptide or
different polypeptides.
 Glycoproteins are proteins with attached sugars.

Many proteins are actually complexes of several polypeptides. The arrangement of more
than a single polypeptide into one protein is termed the quaternary structure of that
protein. Protein complexes might contain two or more copies of the same protein or
they may consist of any number of different polypeptides in various ratios. Such
complexes are certainly not random, but reflect precise interactions among the protein
subunits based on the same sorts of interactions (e.g. hydrophobic, hydrogen bonds, etc)
described above.

Figure 2-16 [47] shows the catabolite activator protein, an example of a protein
containing identical subunits. In this case is has two subunits, so it is termed a dimer.
Each subunit has a cyclic adenosine monophosphate molecule bound that activates the
protein. In the active state this protein binds DNA and activates various genes in the
cell.

Figure 2-16 Catabolite Activator Protein structure

Two depictions of the catabolite activator protein (CAP) of E. coli are shown. The left
panel shows a ribbon depiction, with the two identical subunits shown in red and blue.
CAP senses the presence of cyclic AMP (cAMP) in the cell and a molecule of cAMP
bound to each monomer as indicated. The dimer is actually symmetrical, but that is not
obvious here because the model is rotated slightly. The right panel shows a ball-and-
stick representation of the same dimer and gives a better sense of the overall shape.
Note that the bound cAMP molecules are buried within the protein (so how do they ever
come and go?).

Dinitrogenase is an example of a protein containing non-identical subunits. As shown in

Figure 2-17 [48], the active protein has two copies of one protein (termed α) and two
copies of a second protein (termed β), and therefore is also referred to as a α2β2 tetramer.
This protein is responsible for the reduction of N2 gas to ammonia and is critical to
global nitrogen cycling. The importance of the quaternary structure of dinitrogenase is
only partially clear. The α subunits contain the metal cluster where N2 is actually
reduced (or "fixed") and the β subunit helps form another metal center that helps
transfer electrons to that active site. We can therefore understand why there might need
to be an αβ dimer. However, it is not clear why there are two such αβ dimers hooked
together in the tetramer found in nature. There is, for example, no apparent
communication between the two active sites. Quite possibly, the fact that dinitrogenase
is a tetramer is simply a relic of evolution; perhaps a precursor to dinitrogenase really
did have a need to be a tetramer and there is simply no disadvantage to the protein
retaining that organization.

Figure 2-17 Nitrogenase structure

Nitrogenase is made up of four protein chains, with two copies each of different
proteins, termed the α and β subunits. Though not apparent in this view, the two α
subunits are identical to each other, as are the two β subunits. The quaternary structure
is referred to as an α2β2 tetramer.

Glycoproteins are proteins with attached sugars that are typically important for the
proper function of the protein. In most cases the sugars are added to the protein after it
has been translated. Proteins that encounter the outside environment of the cell are
sometimes glycosylated to stabilize the protein to attack from degradative enzymes and
destructive physical forces. Glycolipids [49] are combinations of lipids and
polysaccharides. In these cases the third glycerol hydroxyl attaches to a polysaccharide
instead of a small polar molecule. These polysaccharides can be quite long, extending
several microns into the outside environment. Lipopolysaccharides [50] (one type of
glycolipid and usually abbreviated LPS) impart several unique properties to the surface
of gram-negative bacteria and we will have more to say about them when examining the
cell wall.

2 - 9 Lipids are the building blocks of membranes

 Membranes have lipids as their major constituent.

 Lipids contain a glycerol backbone. To this backbone are attached a polar group
and two long-chain fatty acids.
Lipids are molecules with two personalities. One part of the molecule wants to associate
with water and the other does not. Molecules with these properties are termed
amphipathic [51]. Figure 2-18 [52] shows that the backbone of the lipid consists of a
three-carbon glycerol molecule. Hydrophhobic, long-chain fatty acids attach to two
hydroxyl groups on the glycerol. To the third hydroxyl group, a polar, and therefore
hydrophilic, group is attached. Many bacteria contain phospholipids in which this third
group contains a phosphate connected to a carbon molecule. The amphipathic nature of
lipids is important in their function in the cell.

Figure 2-18 The structure of phosphatidylethanolamine

The chemical structure (left) and a space-filling model (right) of

phosphatidylethanolamine.

2 - 10 Small molecules are also important in the cell

 NAD and FAD are two common proton and electron carriers in the cell.
 Tetrahydrofolate, cobalamin and coenzyme A are common carbon carriers in the
cell.
 Many enzymes require minerals for proper function in the cell. Common
examples include, iron, zinc, magnesium, and calcium.

There are also a number of important small molecules that shuttle protons, electrons or
small carbon moieties around the cell. These small entities typically do their job in
association with proteins to which they can be either loosely or tightly bound. All of life
on this planet seems to have settled on a surprisingly small set of molecules to perform
these tasks. Almost certainly this is because the use of these molecules evolved early
and has been maintained through evolution.

Proton and electron carriers

Most amino acids are not particularly good at either donating or accepting electrons and
when they do, it is under a limited range of conditions. As you will read in the chapter
on metabolism, the ability to move electrons among proteins is critical to all life, so two
general types of prosthetic group associated with proteins have evolved for this purpose.
Figure 2-19 [53] shows the first type, which are organic multi-ring structures and the
other type are iron-sulfur clusters. In both cases, these carriers have characteristic
affinities for accepting and donating electrons and protons, but these affinities are also
affected by the proteins in which they are found. Thus, a wide range of electron carriers
with different properties has evolved. By organizing these carriers in precise patterns in
the cell, the cell is able to use the transfer of electrons to do work.

Figure 2-19 The structures of a few important electron and hydrogen

carriers

The chemical structures of quinone (left) and nicotinamide adenine dinucleotide (NAD)
(right). In each case, both structures the oxidized and reduced forms are depicted.

Carbon carriers

There are also small molecules in the cell that serve as carriers of important carbon
compounds. Essentially, these carriers have the right chemical properties that make it
relatively easy for enzymes to add or remove a particular carbon unit. Tetrahydrofolate
and cobalamin (vitamin B12) are often involved in adding or removing one-carbon units
during the synthesis of various structures in the cell. Coenzyme A is necessary for the
transfer of small 2 to 4 carbon units (acetyl, propyl) from one enzyme to another. It
finds utility in both the synthesis and breakdown of organic molecules. The beauty of
using a small set of carriers is that it allows the easy movement of carbon from one
pathway to another.

Important minerals
Many types of minerals are important for the proper functioning of enzymes. For
example, magnesium ions are essential for ATP-binding by many enzymes. Zinc is
important in the proper folding of some enzymes and iron, in the form of iron-sulfur
centers and hemes, is critical in many electron transport proteins. Minerals also help
bind structures in the cell together. For example, magnesium and calcium are necessary
for the stabilization of membranes. Potassium ions in the cell shield the large amount of
negative charge on the DNA allowing it to pack more tightly together. More will be said
in later chapters about their specific roles, but some of the more important ions include
K+, PO4-3, Mg+2, Zn+2, Ca+2, Mn+2, Fe+2 and Fe+3.

2 - 11 The cell is organized into functional units

 All cells are organized into functional units. Membranes, cytoplasm, ribosomes
and nuclear regions are found in every cell.

Now that you have had an introduction to the chemicals that make up the typical cell,
we will now look at how this chemistry combines to form major functional units. These
can be thought of as the organizations that carry out the major business of the cell:
growth, replication, feeding and movement. We will first start with membranes because
so many things interact with them. Next internal structures in the cytoplasm will be
described and finally structures outside of the membrane.

First, however, we should describe an important evolutionary hypothesis that will make
sense of much of the following details. As you will see, there are a number of curious
similarities and differences in the details of cellular structure among bacteria, archaea
and eukaryotes. In general much of the machinery in a eukaryotic nucleus [54] and in
the cytoplasm looks rather a lot like what is present in the archaea. However, the
organelles [55] of eukaryotes, such as the mitochondria and chloroplasts, have
properties that are much more similar to those of bacteria. How is this possible? One
clue comes from observing organisms in nature. It is very common to find cooperative
relationships between different species and this is also true in the microbial world. In
some instances these relationships involved close physical contact between their
participants, sometimes with one participant engulfing the other. In 1968 Dr. Lynn
Margulis extended this observation and proposed that some of the organelles found in
eukaryotes, specifically mitochondria and chloroplasts, were originally endosymbionts
of their host. Originally these two microbes probably were able to live independently,
but over time, the endosymbiont lost functionality that its host was already providing
and then became dependent. Over the years ample evidence has accumulated to support
this exceptional insight.

1. Both mitochondria and chloroplasts contain DNA that resembles the

chromosomes of bacteria.
2. Both organelles are surrounded by two membranes reminiscent of gram-negative
cell wall structure (see below) observed in a class of microbes.
3. Mitochondria and chloroplasts divide by a method that resembles binary fission.
4. Much of the internal structure and biochemistry of the photosynthetic organelle
inside chloroplasts is very similar to that observed in cyanobacteria (a
photosynthetic microbe).
5. The ribosomes of mitochondria and chloroplasts resemble those found in
microbes and analysis of the sequence of the 16S rRNA of these ribosomes
 showed that the organelles are in fact closely related to proteobacteria
(mitochondria) and cyanobacteria (chloroplasts) This last bit of proof is very
strongest evidence substantiating Dr. Margulis's hypothesis.

In summary, it seems clear that the eukaryotic cell was born by the merger of an
archaeal cell with a gram-negative proteobacteria. Photosynthetic eukaryotes arose from
a second endosymbiosis, where the eukaryotic cell engulfed a cyanobacterium. Clearly,
eukaryotes, including us, were the result of the cooperation of several bacterial species
in the long distant past.

2 - 12 Membranes surround the cell and hold it in

 Membranes are thin, highly conserved structures found in all cells.

 Membranes are stabilized by hydrophobic interactions, hydrogen bonds and
ionic interactions.
 Membranes are composed of lipids and proteins. The lipids form a bilayer, with
the hydrophilic portion of the lipids facing the aqueous environment, and the
hydrophobic portions clustering together inside the membrane. The majority of
membrane proteins are involved in moving small molecules across the
membrane or in energy generation.

General properties

The cytoplasmic membrane immediately surrounds the inside of the cell and is perhaps
the most conserved structure in living cells. Membranes are thin structures, measuring
about 8 µm thick and every living thing on this planet has some type of membrane They
are the major barrier separating the inside of the cell from the outside and allow cells to
selectively interact with their environment. Membranes are highly organized and
asymmetric. This asymmetry comes from the fact that the membrane that faces the
environment performs very different functions than does the side that faces the
cytoplasm. Membranes are also dynamic, constantly adapting to changing
environmental conditions.

Physical structure

Membranes are composed of lipids and proteins. The majority of lipids are
phospholipids as described earlier, but about 50% of all know bacterial species also
contain hopanoids as shown in Figure 2-75 [56]. These molecules have a similar
structure to sterols found in eukaryotic membranes and serve to help stabilize the
membrane. Proteins are more numerous in bacterial membranes than in eukaryotic
membranes. This is because bacteria in general only contain a single membrane in
contact with the cytoplasm and this has to carry out all the functions of the cell. In
eukaryotes these functions are divided amongst the cytoplasmic membrane and the
other organelles.

Figure 2-75 A hopanoid

The chemical structure and space-filling model of a hopanoid, which is found in many
different bacterial membranes.

Much of the general behavior of membranes is dictated by the behavior of lipids in

water. Because phospholipids are amphipathic, they tend to congregate when placed in
an aqueous environment. This is done in a very specific fashion such that the
hydrophilic portions face the water and the hydrophobic portions are buried inside.
Under the cell's direction lipids are organized into a bilayer, where there are two sheets
of lipids oriented so that the hydrophobic faces of each sheet face each other as shown
in Figure 2-20 [57]. Lipid bilayers can be almost any size and can form vesicles
spontaneously, if lipids are placed in an aqueous environment. In the cell, however, the
synthesis of membranes is performed by specific enzymes and is tightly controlled.

Figure 2-20 A model of a lipid bilayer

A space-filling representation of a lipid bilayer as developed by H. Heller et al. (J. Phys.
Chem. 1993. 97:8343-60).

The cytoplasmic membrane is held together by a number of forces. Hydrophobic

interactions between the alkyl chains of neighboring lipids are a major component of
membrane stability. Hydrogen bonds between lipids and between membrane proteins
and lipids also hold a membrane together. Further stability comes from negative charges
on proteins that form ionic interactions with divalent cations such as Mg+2 and Ca+2 and
the hydrophilic head of lipids.

The hydrophobic region of the membrane provides a critical function: it prevents polar
compounds, such as ions and most biological molecules, from passing through it. This
allows the cell to create and maintain gradients of ions and small molecules across the
membrane by mechanisms described below.

So how do polar molecules ever cross this membrane, since this represents an important
interaction between the cell and its environment? This transfer across the membrane
comes about through the specific functioning of proteins that are imbedded in the
membrane. Some proteins span the membrane while others are exposed on the outside
or the inside. These proteins may move within the plane of the membrane or they may
be anchored to structures in or near the membrane. Many of the membrane-spanning
proteins are involved in transport of the polar molecules that must pass through the
membranes. A subset of these proteins are also involved in energy generation as
discussed below.

The membrane is fluid and has the consistency of a light grade oil. It has been termed a
fluid mosaic: "fluid" because the lipids are free to move about on each side of the
membrane and "mosaic" because there is a definite pattern to it. Lipids do not generally
switch from outside to inside or vice versa, because of the problem of trying to move
the hydrophilic group through the hydrophobic core of the bilayer.

2 - 13 Membranes are a selective barrier

 Membranes are semi-permeable structures that prevent most common biological

compounds from moving across them.
 Biological compounds can be moved across the membrane by facilitated
diffusion, group translocation, and active transport. All of these functions are
mediated by proteins in the membrane.

The concentration of solutes, sugars, and most ions is generally much higher within the
cell than outside. A fundamental principle of nature is that different concentrations of a
given solute tend to equilibrate across a boundary due to diffusion [58]. However, the
cell boundary is the membrane and its hydrophobic core prevents this diffusion for polar
molecules. Compounds such as amino acids, organic acids and inorganic salts must
therefore be specifically transported across the membrane by proteins and once inside
these molecules cannot escape. The cell can therefore control the nature and amount of
these compounds that enter or leave the cell.

One of the basic ingredients of life is water, and this must be present inside the cell for
it to function. Water can diffuse relatively slowly across the membrane, but it was a
matter of debate whether this was fast enough for cellular processes. Studies of
adaptation to osmotic stress suggested that passive diffusion of water was too slow to
explain the rapid changes observed in some bacteria. A search began for the elusive
protein that was carrying out this process. The first water channel was discovered by
Peter Agre in 1988. These proteins were first decsribed in eukaryotic cells, but a wide
variety of living systems are now know to contain them and the term aquaporins was
coined to describe them. Over 100 aquaporins have been discovered in bacterial systems
and while these do not share a high degree of sequence homology with eukaryotic
aquaporins, they share significant structural similarity. Aquaporins can be divided into
two large classes, those that only allow water to flow through them, and those that will
also transport glycerol and a few other small uncharged molecules. Aquaporins have not
been found in every microorganism, in fact some species appear to lack them. In these
cases the microbe may rely solely on diffusion across the membrane, or its aquaporin
has a novel sequence.

Aquaporins (Figure 2-76) are intrinsic membrane proteins of about 23 kDA that contain
6 α-helices arranged around a central core channel. Each aquaporin will associate with
three others to form a homotetramer in the membrane. The central channel is narrow
and contains a conserved stucture that can selectively allow only the passage of the
desired molecule. In this way, water can rapidly flow into and out of the cell, speeding
up its transport through the membrane.
Figure 2-76 Aquaporin

A ribbon diagram of the 23 kDA Aquaporin from E. coli. Each of the 6 α helices that
line the water channel are shown. The arrows indicate the path of water through the
protein. The two sequences of 3 amino acids (Asparagine-Proline-Alanine) are shown in
pink. This area is thought to be important in discriminating, only allowing water
molecules to pass through the pore. Thus, serving as a gate.

While aquaporins are essential to cell function they also create a serious problem. The
inside of the cell is full of many types of solutes: proteins, nucleic acids, other small
molecules and ions. In comparison the outside environment, in most cases, is very
dilute. Because of this there is a higher concentration of water outside the cell than
inside the cell. Nature hates imbalances such as this and in an effort to correct the
problem; water tends to flow into the cell, by a process called osmosis [59]. Osmosis
causes a high pressure against the cell membrane. This pressure would rapidly cause
lysis of most cells and one of the major purposes of the peptidoglycan of the cell wall
(discussed below) is to prevent the cell membrane from bursting.

For molecules that are soluble in both the lipid membrane and the surrounding aqueous
environment, the law of simple diffusion directs transport. The membrane is not a
barrier for such molecules. These types of molecules are uncommon since solubility in
both a hydrophobic and a hydrophilic environment is unusual. There is no transport
protein for such compounds, so there is no specificity of control or energy cost. The cell
cannot create a concentration gradient of these molecules. One important example is
water. Water can pass freely into and out of cells.

There are three basic types of transport systems

  Facilitated Diffusion
 Group Translocation
 Active Transport

Many of the proteins in the membrane function to help carry out selective transport,
particularly of polar compounds. These proteins typically span the entire membrane,
making contact with the outside environment and the cytoplasm. They often require the
expenditure of energy to help compounds move across the membrane, though cells can
also use concentration gradients of these compounds to generate energy, as described
below

Facilitated diffusion

This process involves a protein that binds the molecule to be transported and physically
moves that compound through the membrane. Binding of the molecule to the protein
causes a conformational change in the protein so that the molecule now faces the
opposite side from where it was. Facilitated diffusion, as shown in Figure 2-21 [60], is
therefore specific because a protein must bind the molecule. However, these small
molecules are readily moved in and out of the cell, so a gradient cannot be formed nor is
energy required. One example of a protein involved in facilitated diffusion is the
glycerol facilitator protein. In E. coli this enzyme binds glycerol and a few other
polyalcohols and allows their diffusion into the cell. Once inside, the glycerol is
immediately phosphorylated, preventing its diffusion back outside the cell.

Figure 2-21 Facilitated Difusion

An animation of the migration of solutes in and out of the cell as facilitated by a protein.
Notice that this mechanism does not lead to a solute concentration inside the cell that is
higher than outside. Rather, it leads to an equilibrium of that solute across the gradient.

Group translocation

In this process, a protein specifically binds the target molecule and transports it inside
the cell while simultaneously modifying it chemically. Most group translocations
require energy and tend to be unidirectional, unlike facilitated diffusion. The substrates
of catabolic pathways, such as sugars, are sometimes transported by group translocation.
This is an efficient way to both bring substrate into the cell and begin the breakdown
process. Figure 2-22 [61] shows an animation of group translocation.

Figure 2-22 Group Translocation

An animation of group translocation. The glucose molecule that is being transported

into the cell is modified by the addition of a phosphate from phosphoenolpyruvate to
form glucose-6-phosphate.

Active transport

In active transport, energy is expended to transport the small molecules, but they are not
chemically altered. The process is efficient enough to cause the internal concentration in
the cell to reach many times its external concentration. Active transport proteins are
molecular pumps that expend energy to pump their substrates against a concentration
gradient. This energy comes in two forms: ATP and ion gradients (both ATP and ion
gradients are made by central metabolism and we will cover their formation in the
chapters on metabolism). In ATP-based active transport, ATP hydrolysis is coupled to
the movement of the small molecule across the membrane. One large group of proteins
involved in this type of transport is the ATP binding cassette (ABC) transporters. ABC
transporters have been found in all living species with 80 identified in the E. coli
genome and 48 in the human genome. The mechanism of ABC transporters is
exemplified by the maltose binding protein of E. coli.

Figure 2-23 Active Transport

Three separate types of transport are shown. In the first, an antiporter moves two
different small molecules across the cell membrane, but in the opposite directions. In
the second, a symporter moves two or more different molecules to move into the cell
simultaneously. Typically, the desired molecule is being concentrated against a gradient
and that transport is driven by the transport of the other molecule, which is moving with
a gradient. Finally, a uniporter binds and transports the target molecule only. Energy is
required for these processes and the cell can accumulate molecules inside the cell using
this mechanism.

Ion gradient active transport uses the energy of one chemical gradient, that of the
specific ion, to drive the creation of a different gradient, the uptake of the small
molecule. The ion gradient that supports the work is at a higher concentration on one
side of the membrane than the other. The transport protein both binds its small molecule
to transport and provides a gateway for this ion to fall down its concentration gradient.
When the ion moves through its gateway, it causes a conformational change in the
protein and this change is used to transport the target small molecule into the cell.

Active transport proteins may be highly specific for only one molecule or may be able
to carry a class of chemically related molecules. The animation in Figure 2-23 [62]
shows several different types of transport molecules. An example of a more general
transport protein is the branch chain amino acid transporter of Pseudomonas
aeruginosa, which transports leucine, valine, and isoleucine. Figure 2-63 [63]
summarizes the various properties of transport mechanisms.

Figure 2-63 Properties of various transport systems

Passive Facilitated Active Group

Property
Diffusion Diffusion Transport Translocation
Carrier Mediated - + + +
Concentration Against
- - + Not Applicable
Gradient
Specificity - + + +
Energy Expended - - + +
Solute Modified During
- - - +
Transport
A comparison of the methods for transporting molecules through the membrane and
into the cell.

2 - 14 Membranes can help generate energy

 In many microorganisms a collection of proteins are involved in generating

energy. These proteins move high energy electrons down an electron transport
chain and in the process move protons from the inside to the outside of the
membrane.
 This proton gradient can do work by driving other proteins that form ATP, that
transport molecules across the membrane, or that move the cell.

Many cells use respiratory processes to obtain their energy. During respiration, organic
or inorganic compounds that contain energy are oxidized, releasing electrons to do
work. In many microorganisms these electrons find their way to the membrane where
they are passed down a series of electron carriers as shown in Figure 2-24 [64]. During
this operation, protons are transported outside the cell. This creates a gradient of protons
across the cell membrane, energizing it, in a fashion similar to charging a battery. The
energy of this gradient can then be used to do work directly, a process known as the
proton motive force, or can be channeled into a special protein known as ATP synthase.
ATP synthase can convert: ADP to ATP, and the ATP can itself do work.

Figure 2-24 Generating the proton motive force

Membranes are critical in many cells for the generation of usable energy. The cartoon
shows the various membrane proteins involved in converting high-energy electrons
from photoreceptors into useful energy. They do so by forming a proton gradient across
the cell membrane, termed a proton motive force, which is in turn used by other proteins
to synthesize ATP. This will be discussed in greater detail in the chapter on metabolism.

The prokaryotic cells performing photosynthesis have membrane systems specific to

that process. Light excites electrons found in pigmented proteins in the membrane and
the electrons are again passed down through a series of electron carriers. As above, a
proton motive force is generated and used to synthesize ATP. The specifics of these
systems are discussed in the chapter on metabolism.
2 - 15 Membranes are also important in their own synthesis and can fold
inward for specialized functions

 Membranes also contain some of the enzymes that are necessary for membrane
synthesis.
 Some functions that occur in the membrane require large amounts of surface
area. Membranes can fold inward, into the cytoplasm to provide the extra needed
space.

Membranes also contain specialized enzymes that carry out certain biosynthetic
functions. For example, the last few steps of lipid synthesis take place inside the
membrane. Another example is cell wall synthesis and assembly. Much of the synthesis
of cell wall monomers occurs there and the stitching together of the cell wall polymer
takes places while it is anchored to the membrane. In addition, any cellular protein that
carries out its function outside the cell membrane (such as outer membrane and
extracellular proteins) must pass through that membrane. During their synthesis the
ribosome is guided to the cytoplasmic face of the membrane and the growing peptide
chain is synthesized directly into the lipid bilayer. Integral membrane proteins then fold
up and stay in the membrane while extracellular proteins move through the membrane
and take on their final shape on the other side.

Infoldings of the membrane are found in some photosynthetic bacteria. These bacteria
use pigments in their membranes to capture light energy. Under low light, they need to
increase the surface area to catch more light. They cannot make the membrane thicker,
but they can increase the surface area by creating regions where the membrane folds
into the cytoplasm. These invaginations are still attached to the cytoplasmic membrane
and a picture of such structures, termed the intracytoplasmic membrane in the case of
Rhodobacter sphaeroides, is shown in Figure 2-25 [65].

Figure 2-25 The intracytoplasmic membrane of Rhodobacter sphaeroides

This electron micrograph shows the complicated infolding of the cytoplasmic
membrane of R. sphaeroides when it is performing photosynthesis. This infolding
creates a larger membrane surface area into which light-harvesting complexes can be
inserted. Under low-light conditions many light-harvesting complexes are needed to
capture the small number of photons striking the microbe. (Source: Samuel Kaplan,
University of Texas - Houston Medical School)

2 - 16 The cytoplasm is the area inside the membrane

 The prokaryotic cytoplasm, while visually unadorned, is structured and is the

site of most of the important metabolic reactions in the cell.
 There is little free water in the cytoplasm. This gives it a gel-like consistency.

The cytoplasm or protoplasm [66] is the portion of the cell that lies within the
cytoplasmic membrane. The cytoplasmic matrix is defined as substances within this
membrane, excluding the genetic material. In most prokaryotes, it appears to be
relatively featureless by electron microscope, but that simply means that there are no
large structures in the matrix. This is in contrast to eukaryotic cells, which have
mitochondria [67], and typically other visible organelles [68] that exist for different
specific functions. Despite this visual simplicity, the prokaryotic cytoplasm is the site of
almost all of the important metabolic functions in the cell.

The cytoplasm has a gel-like consistency, with rather different properties than the
simple solutions that we typically make up in the laboratory. This is because there is
surprisingly little free water in the cell. Rather than picturing the cytoplasm as a pool of
water with the occasional large molecule floating around, it is better to think of it as a
bag of proteins and other macromolecules, each coated with a layer of water, and with a
modest number of free water molecules bouncing around in between. In fact, there is so
little free water in the cell that one-third of all water molecules are making hydrophilic
contacts with the macromolecules in the cell. Given this difference between our lab
solutions and the actual nature of the cytoplasm, it is a bit surprising that the
biochemical analyses we perform in the lab mimic the behavior observed in the cell.

2 - 17 Enzymes serve as catalysts in the cytoplasm

 The cytoplasm can be highly structured. One example is the complex assembly
of enzymes that perform DNA replication.

The cytoplasm is the site of the majority of metabolism of the cell and the details of
these processes are covered in the chapters on metabolism and photosynthesis.
However, there are a few general issues concerning enzymes in the cytoplasm that
should be mentioned here.

Though the cytoplasm appears featureless, there is actually a significant amount of local
organization. A good illustration of this can be found by examining the enzymes of
DNA replication. Though too small to be seen, the proteins that perform replication are
in complex assemblies of many proteins. This is much more efficient that having each
protein float around, and simply finding the right time and place to perform its function
by random chance. For example, DNA gyrase, which unwinds and opens the DNA for
copying, has to function in coordination with DNA polymerase, which inserts each new
nucleotide in the growing strand. Without this coordination, the DNA would not open
up for replication and the process would simply not occur. In some other processes it
would certainly be possible for the enzymes to float around in the cytoplasm without
any interaction, but often it is much more efficient to organize in some fashion. The
glycolytic enzymes (enzymes that oxidize sugars for energy) are an example of this type
of multi-enzyme complex. One enzyme directly hands over its product to the next
enzyme for which it is the substrate - a sort of molecular assembly line. This is much
more efficient, because substrate does not accumulate where it should not and the local
concentration of substrate for each enzyme is very high.

2 - 18 The cell DNA is organized into a nucleoid

 Prokaryotic DNA is organized into a DNA-protein complex called the nucleoid.

 The chromosome is much longer than the typical prokaryote and must be
compacted to fit inside the cell. This is accomplished by supercoiling the DNA
(adding twists) and complexing it with DNA-binding proteins.

The nucleoid [69] is a bit difficult to define because unlike eukaryotes with their nuclei,
prokaryotic chromosomes are not confined to a specific organelle. The nucleoid is
sometimes referred to as the region of the bacterial cell that contains the chromosome,
but that suggests there is a specific region in any cell where the chromosomes is always
found, and this is probably not true. A better definition might be that the nucleoid is the
region of the cell that is currently occupied by the DNA-protein complex that makes up
the chromosome. So what do we know about prokaryotic chromosomes? What does a
typical chromosome look like?

It is a little awkward to talk about "typical" anything in prokaryotes, since there is

always such a large range for any given property. In the case of prokaryotic
chromosomes, the smallest known is about 160 kB, while the largest is about 10,000
kB. E. coli is certainly well-studied and might well be considered typical, with strain
K12 having a chromosome of 4,639,221 bp containing 4405 genes. The chromosome
for this bacterium is circular and this is a common arrangement, but there are a number
of species with linear chromosomes. If stretched out, this material would be about 1400
µm long or about 1/16th of an inch. The E. coli cell is about 1-5 µm in length so it is
clear that an amazing degree of packing is necessary to fit the chromosome inside its
tiny host. The nucleoid occupies about half of the bacterial cytoplasm and has a density
of 20-50 mg/ml, similar to what is observed for the nucleus of a non-dividing eukaryotic
cell. The following description of the nucleoid refers specifically to that of E. coli, but is
probably generally similar to that of other prokaryotes. Figure 2-26 [70] shows the
nucleoid of E. coli.

Figure 2-26 Electron micrograph of the nucleoid

The nucleoid as seen in thin sections of growing E. coli. Panels A and B show the same
section; in panel B the ribosome-free spaces were enhanced by coloring by hand. Panel
C shows a similar cell stained with antibodies specific for DNA. (Source: E.
Kellenberger).

The nucleoid is composed of DNA in association with a number of DNA-binding

proteins that help it maintain its structure. The protein HU non-specifically binds to
DNA and bends it, with the DNA apparently wrapping around the HU protein. Another
protein found on DNA is IHF, whose sequence is reminiscent of that of HU and is
therefore evolutionarily related. It also facilitates bending of DNA, but it does so by
binding to specific DNA sites. A third protein, H-NS, binds to DNA non-specifically
and is apparently involved in compacting DNA structure. It is found throughout the
nucleoid and is likely the major DNA-binding protein that organizes the chromosome.
The nucleoid also contains a large amount of RNA polymerase and RNA, as well as
small amounts of many different proteins that regulate the expression of specific genes.
These seem not to perform any structural role, but reflect the importance of RNA
transcription in the nucleoid in growing cells.

The DNA double helix typically has a bit of twisting tension in the opposite direction of
the helix ladder (the twists typically "unwind" the left-handed DNA helix). Negative
supercoiling, as it is called, is produced by the action of enzymes termed
topoisomerases. Figure 2-27 [71] shows supercoiling of the DNA. This negative
supercoiling makes it slightly easier to separate the two strands of the double helix, as
must be done to start transcription and replication.

Figure 2-27 Supercoiling of DNA strands

Supercoiling is very difficult to show in a static picture and it is hard to create a cartoon
that adequately describes it. Instead consider this: Take a few feet of tubing or a garden
hose and hold one end in your left hand. Then with your right hand, rotate the other end
along its long axis. Since the tubing/hose is resistant to this twisting, this energy will
cause a section of the tubing to form a new structure, a bit like the middle picture above.
This "supertwisting" is supercoiling. Now the analogy between tubing and a DNA
double-helix is not bad - both objects are able to bend but cannot really rotate much
along their long axis, so if one could do the same thing with a piece of DNA, the same
supertwisting would result. Of course no one is "holding one end of the DNA" in the
cell, but the fact that the DNA is a closed circle has the same effect. In this picture,
double-stranded DNA is shown, but for clarity the helical nature of the DNA is not
depicted (though it certainly IS helical). Twist refers to the number of helical turns in
the DNA and writhe to the number of times the double helix crosses over on itself
(these are the supercoils). The bottom two figures shows how the supercoils could be
concentrated in a single region or dispersed.

As shown in Figure 2-28 [72], the chromosome is further folded into 50 or so loops of
about 100,000 base pairs. These domains supercoil independently and indeed, even
small sections within the same loop can transiently have different degrees of
supercoiling. Because supercoiling changes fairly rapidly, it has been extremely difficult
to study in living cells. Nevertheless, it is clear that a variety of environmental effects
and even the act of transcription itself can affect the supercoiling of a region. In turn, the
specific supercoiling of a region can affect the ability of the cell to express genes in that
region. Because supercoiling is affected by so many things and in turn can affect
expression of much of the genome, it will be important for microbiologists to develop a
better understanding of this phenomenon in the future.

Figure 2-28 Chromosome structural organization

A model of the overall structure of the bacterial chromosome. (A) The unfolded,
circular chromosome of E. coli depicted as a single line for simplicity, though of course
it is a double-stranded helix. (B) The DNA folded into chromosomal domains by
protein-DNA associations. The proteins are depicted as the black circles, interacting
with both the DNA and with each other. Six domains are shown, but the actual number
for E. coli is about 50. (C) Supercoiling and other interactions cause the chromosome to
compact greatly.

2 - 19 Transcription and translation occur on the surface of the nucleoid

So we have told you that the DNA in the cell has a very compact structure because of
DNA-binding proteins, but we have also said that that structure is very dynamic because
gene transcription is going on all of the time. This paradox creates a very difficult
situation for the cell that only becomes worse when it is time to replicate the
chromosome. Remember that prokaryotes continue to perform gene expression
throughout replication, in contrast to eukaryotes. As you will learn below, transcription
and translation are coupled in bacteria - the beginning of the messenger RNA (mRNA),
termed the 5' end, is being actively translated while last portion, termed the 3' end, is
still being synthesized. This process is depicted in Figure 2-29 [73]. Yet the internal
regions of the nucleoid appear to be devoid of ribosomes and non DNA-binding
proteins, suggesting that all transcription and translation must occur on the surface of
the nucleoid. Therefore the cell must shift this large tangle of DNA around by some
unknown mechanism as gene expression of certain buried sequences is needed. It is a
marvel that the whole process of transcription, translation and replication works at all,
especially within the very small confines of the cell.

Figure 2-29 The coupling of transcription and translation

In prokaryotes the process of transcription and translation are tightly coupled. This
increases the rate at which proteins can be expressed and is one reason that some
bacteria can multiply so quickly.

2 - 20 Translation involves messenger RNA

 Most messenger RNAs do not contain a great deal of secondary or tertiary

structure.
 Messenger RNA is normally unstable with half-lives of less than three minutes.
 Prokaryotic messenger RNA has short stretches of adenine added to the 3' end of
the message. This poly-A tail serves as a target for degradation.
Translation is the process of converting the instructions coded in the DNA into the
proteins that actually carry out the work. The macromolecules that perform this task
consist of mRNA, transfer RNA [74] (tRNA) and the ribosome, which is made of
ribosomal RNA [75] (rRNA) and ribosomal proteins. In brief, this process consists of
making an mRNA copy of a region in the DNA that gives directions for the synthesis of
a protein or proteins. The mRNA is then bound by a ribosome that translates the mRNA
into a amino acid sequence. The amino acids necessary for the protein are carried to the
ribosome by tRNA that actually read the information in the mRNA and add the
appropriate amino acid to the nascent protein chain. This is described in much greater
detail in the chapter on the central dogma.

We will now examine the structure of the molecules involved in translation, starting
with mRNA, where the primary structure is simple - merely unmodified A, G, C and U
bases. In almost all prokaryotic mRNAs there is not a great deal of secondary and
tertiary structure, since they are typically being translated by ribosomes and the
translating ribosome certainly removes any structure as it moves along the mRNA.
What structure there is tends to be in the regions that are not translated, notably the 5'
and 3' ends of the mRNA. One of the roles of structure, especially at the 3' end, is to
stabilize the mRNA. Now it happens that most prokaryotic mRNAs are not very stable
in the cell because they are rapidly degraded by RNases. However, different types of
RNA structures can impede the progress of nucleases, particularly the type that
degrades from the 3' end of the mRNA (termed exonucleases, because they attack from
the exterior ends). As a complication, however, there are some RNA structures that
actually serve as a specific target for other types of RNases (termed endonucleases,
because they cut within an RNA) and thus lead to destabilization of the mRNA. This is
biologically important because a more stable mRNA is translated by more ribosomes
and therefore leads to more protein product.

As a final structural feature, most if not all prokaryotic mRNAs have short stretches of
adenosine residues added to the 3' end after transcription. The presence of these also
tends to lead to mRNA degradation. This is a bit surprising because the presence of long
adenosine stretches on the 3' ends of eukaryotic mRNAs actually tends to stabilize those
mRNAs. This appears to be a case where evolution has taken a single feature, addition
of adenosines to mRNAs, and changed its functional importance through evolution.

In eukaryotes, the fact that mRNA is transcribed in the nucleus and must be exported to
the cytoplasm for translation changes some details. One of these is that eukaryotic
mRNAs are rather more stable than in prokaryotes.

2 - 21 Ribosomes are composed of RNA and protein

 Ribosomes are 62% RNA and 38% protein formed into two complexes in
prokaryotes, the 50S ribosome subunit and the 30S ribosome subunit.
 Ribosome associated proteins are positively charged. This helps them to bind to
the negatively charged mRNA.

In contrast to the case with mRNAs, the other RNAs involved in translation, tRNA and
rRNA, have very distinct structures. Each rRNA folds into a known secondary structure
and has a complex tertiary structure containing many short helical regions and long
range base pair interactions. These structures are also maintained by interactions
between the RNAs and protein.

The composition of ribosomes is 62 % RNA and 38 % protein by weight. Two

complexes of RNA and protein make up the ribosome, the 30S subunit and the 50S
subunit. (The S stands for Svedberg [76] units, a measure of how fast something
sediments in solution. For our purposes all you need to know is that larger molecules
sediment faster and have larger Svedberg units. The unit is named after Theodor
Svedberg who won the Nobel Prize in Chemistry in 1926 for his work on suspensions of
large molecules and other compounds in solutions.) The 30S subunit is composed of 21
proteins and a single-stranded rRNA molecule of about 1,500 nucleotides, termed the
16S rRNA. The 50S subunit contains 31 proteins and two RNA species, a 5S rRNA of
150 nucleotides and a 23S rRNA of about 2,900 nucleotides. Several of the nucleotides
on the 16S and 23S rRNAs have been modified by methylation and these modifications
are probably critical to the function of the rRNAs since they always happen in regions
conserved through evolution.

The ribosome-associated proteins are positively charged, with a high proportion of

lysine and arginine residues. This facilitates complex formation between the acidic
RNA and these basic proteins. The crystal structure of the entire 70S ribosome has been
solved as shown in Figure 2-30 [77], which is quite remarkable because solving such
structures becomes more difficult as the size of the complex increases and the ribosome
is huge. We are still trying to fully understand what that structure tells us about
ribosome function. We also know that the ribosome is not static, but dynamic, changing
shape during a translation cycle. It is also clear that this complex mix of protein and
rRNAs can self-assemble when mixed in the right order, though the process of assembly
in the cell is certainly more carefully controlled.

Figure 2-30 The crystal structure of the 70S ribosome

A molecular model of the 70S ribosome of Thermus thermophilis showing the position
of proteins (ball-and stick) and RNA (Gray and blue-green ribbons) within the structure.
Note the prominence of RNA in the ribosome, which constitutes over 60% of the
molecular weight. (Source: adaption from S. Petry et al. 2005. Cell 123:1255-1266)

2 - 22 Transfer RNA is the ferry for amino acids

 Transfer RNA has an L shape in its native three-dimensional form.

 There are unique tRNAs for each of the 20 common amino acids
 Aminoacyl-tRNA synthetases recognize their respective tRNA and add the
appropriate amino acid.

Transfer RNA (tRNA) is the ferry that transports the amino acids to the ribosome. There
are one or more different tRNA molecules for each of the 20 amino acids. Each consists
of 70 to 80 nucleotides of single-stranded RNA that is extensively base-paired to form
four short helical domains. These structures are commonly represented as a two-
dimensional cloverleaf, but look more like an "L" in the native three-dimensional
structure as shown in Figure 2-31 [78]. Now the tertiary structures of tRNAs are all
rather similar, so the critical features that make each appropriate to a specific amino
acid are largely found in the primary structure itself. Many bases in tRNA molecules are
chemically modified by enzymes to help the molecule carry out its function.

Figure 2-31 The structure of tRNA

The two-dimensional structure of tRNA looks like a cloverleaf, but in the actual three-
dimensional form, it has a surprising L-shaped structure as shown. The bottom of the
structure as shown here contains the anticodon that interacts with the mRNA. The top
right of the structure is where the appropriate amino acid residue is attached by
synthetases.

The aminoacyl-tRNA synthetases are the enzymes that add the amino acid to the
tRNAs. Figure 2-32 [79] depicts a complex between an aminoacyl tRNA synthetase and
tRNA. There is a single synthetase for each amino acid and it binds each of its
appropriate tRNA molecules and charges it with its appropriate amino acid. The
synthetases avoid both the wrong tRNAs as well as the wrong amino acids. However,
the process is somewhat trickier than it first appears. First, one might expect that the
synthetase might recognize the proper tRNAs by examining the anticodon [80] loop, the
part recognized by the ribosome to match the tRNA to the mRNA. After all, the
anticodon certainly does define the amino acid in translation. This is not the case,
however, perhaps because the anticodon is very far away from the end of the tRNA that
is charged with the amino acid. In any event, it is clear that most of the basis for proper
synthetase-tRNA recognition lies elsewhere in the tRNA. This then raises another
problem: when one looks at the different tRNAs that all carry a given amino acid and
are therefore all recognized by a single synthetase, no obvious pattern emerges. In other
words, the important features that allow the alanine synthetase to recognize only alanine
tRNAs are not completely clear, though great strides in understanding this process have
been made.

Figure 2-32 The amino acyl-tRNA synthetase complex

A molecular model of aminoacyl tRNA synthetase binding its tRNA. The tRNA is
shown as ball-and-stick, while the synthetase is depicted in the ribbon form. In this
picture, the anticodon is at the top of the figure and the site of amino acid attachment is
at the bottom right. Note that the synthetase does not "sense" the anticodon directly.

One final thought before we move on. Think about the chicken-and-egg conundrum that
translation brings up. This whole process has the express purpose of synthesizing
proteins, yet it involves proteins at every step. How could these proteins have evolved to
serve this function when they are needed for their own synthesis? In other words, how
could you synthesize any protein until a complete set of translation proteins had
evolved? Part of the answer is that primordial translation was probably much simpler,
though less accurate and efficient. Perhaps only very few and somewhat general protein
functions were actually required. Alternatively, perhaps early translation used no
proteins at all: Some scientists believe that early life employed RNA molecules that
were capable of both carrying out necessary enzymatic functions and storing hereditary
information. They posit that it was only later that proteins came along and started to
assist in their own synthesis. However, this hypothesis still does not explain how one
simultaneously evolved functional proteins and a process for creating them.

2 - 23 Inclusions and other internal structures are found in many

prokaryotic cells

 Prokaryotic cells do have some microscopically visible structures in their

cytoplasm. They serve specific purposes for the cell.
  Inclusions are aggregates of specific chemical compounds and often serve as a
reservoir of energy or carbon. Common inclusions are: poly-β-
hydroxyalkanoate, sulfur globules, and polyphosphate.
 Gas vesicles are found in many aquatic microorganisms are serve to adjust the
location of the microbe in the water column.
 Magnetosomes are structures that sense the magnetic field of the earth. Cells
often use these to orient themselves appropriately in their environment.

We said before that there were few structures in the prokaryotic cytoplasm that are
visible by microscopy. Some of those that are seen are discussed below. In general, they
serve specific purposes in the cell and are often found only in certain cell types or under
certain growth conditions.

Inclusions

Inclusions are dense aggregates of specific chemical compounds in the cell. Typically,
the aggregated chemical serves as a reservoir of either energy-rich compounds or
building blocks for the cell. Forming polymers costs energy and it may seem wiser for
the cell to keep the excess monomers around for when they are needed. The benefit of
polymerization is that it decreases the osmotic pressure on the cell, a serious problem as
described later. Inclusions often accumulate under laboratory conditions when a cell is
grown in the presence of excess nutrients. However, the role of some inclusions is
unclear. Growth on rich medium causes their creation, but subsequent starvation in the
test tube does not always result in the use of these reserves. This suggests that these
inclusions, at least, are not storage bodies.

Poly-β-hydroxyalkanoate

One of the more common storage inclusions involves poly-β-hydroxyalkanoate (PHA).

It is a long polymer of repeating hydrophobic units that can have various carbon chains
attached to it. The most common form of this class of polymers is poly-β-
hydroxybutyrate, which has a methyl group as the side chain to the molecule as shown
in Figure 2-33 [81]. The function of PHA in bacteria is as a carbon and energy storage
product. Just as we store fat, some bacteria store PHA. Some PHA polymers have
plastic-like qualities and there is some interest in exploiting them as a form of
biodegradable plastic.

Figure 2-33 Poly-β-hydroxyalkanoate inclusion bodies

The figure shows an electron micrograph of inclusion bodies of poly-β-

hydroxyalkanoate inside a cell of a Rhodobacter sphaeroides. The specific chemical
here is poly-β-hydroxybutyrate (PHB). In the generalized poly-β-hydroxyalkanoate
structure shown at the left, the R group is a methyl in PHB. (Source, Sam Kaplan,
University of Texas - Houston Medical School)

Glycogen

Glycogen is another common carbon and energy storage product. Humans also
synthesize and utilize glycogen, which is a polymer of repeating glucose units.

Phosphate granules and sulfur globules

Given the opportunity, many organisms accumulate granules containing long chains of
phosphate, since this is often a limiting nutrient in the environment. These
polyphosphate polymers, also called volutin, form visible granules in some microbes.
These granules are readily stained by many basic dyes such as toluidine blue and turn
reddish violet in color. These inclusions are often referred to as metachromatic granules
because they become visible by "metachromasy" (a color change). Polyphosphate is
found in all known cells (eukaryotes, bacteria and archaea) and appears to serve many
important roles.

1. It serves as a phosphate reservoir

2. It is an alternative substrate in place of ATP when phosphorylating sugars
during catabolism.
3. It is a chelator for divalent cations
4. It can be a buffer under alkaline stress
5. It is an important factor for DNA uptake.
6. Finally, phosphate polymers are important regulators in response to stress

Figure 2-34 [82] depicts another visible structure, termed a sulfur globule, which is
found in a variety of bacteria capable of oxidizing reduced sulfur compounds such as
hydrogen sulfide and thiosulfate. Oxidation of these compounds is linked either to
energy metabolism or photosynthesis. Oxidation of sulfide initially yields elemental
sulfur, which accumulates in globules inside or outside the cell. If the sulfide is
exhausted the sulfur may be further oxidized to sulfate.

Figure 2-34 Sulfur globules

Sulfur globules found in Thiomargarita namibiensis. This large microbe (100 to 750
µm in size) is found in Walvis Bay off the coast of Namibia. (Source: copyright Max
Planck Institute for Marine Microbiology, Bremen, Germany)

Gas vesicles
Figure 2-35 [83] shows an example of gas vesicles [84], also known as gas vacuoles, that
are found in cyanobacteria. Cyanobacteria are photosynthetic and live in lakes and
oceans. In these environments, the cyanobacteria use gas vesicles to control their
position in the water column to obtain the optimum amount of light and nutrients.

Figure 2-35 Gas vesicles

The hexagonal forms inside the cytoplasm of this cyanobacterium are the gas vesicles.
These actively dividing cells are Microscystis sp.(Source: A. E. Walsby, 1994.
Microbiol. Rev. 58:94-144)

Gas vesicles are often aggregates of hollow cylindrical structures composed of rigid
proteins. They are impermeable to water, but permeable to gas. The amount of gas in
the vesicle is under the control of the microorganism. Release of gas from the cell
causes the bacteria to fall in the water column, while filling the vesicle with gas causes
the cells to rise.

Magnetosomes

magnetosomes [85] are intracellular crystals of iron magnetite (Fe3O4) that impart a
permanent magnetic dipole to prokaryotic cells that have them. They allow these
microbes to orient themselves in a magnetic field. This process does not appear to
involve any special machinery beside the magnetosome, Each microbe can be thought
of as having a tiny magnet that is responding to the magnetic field in the environment.
These magnetosomes allow the microbes to follow the magnetic field of the earth. Some
species of magnetotatic bacteria have the following behavior. In the northern
hemisphere magnetotatic bacteria swim north along the magnetic field, while in the
southern hemisphere they swim south. Because of the inclination of the earth's magnetic
field, this causes the microbes to swim downward. Many microbes containing
magnetosomes are aquatic organisms that do not grow well in the presence of
atmospheric concentrations of oxygen and they move away from the oxygen higher up
in the water column by detecting the magnetic field and swimming downward.

A special membrane surrounds magnetosomes that confines the magnetite to a defined

area. The membrane likely plays a role in precipitating the iron as Fe3O4 in the
developing magnetosome. Magnetosomes can be square, rectangular or even spike-
shaped. Magnetosomes are primarily found in aquatic bacteria and in some unicellular
algae (eukaryotes).

2 - 24 The periplasm is between the cytoplasmic and outer membranes in

gram-negative bacteria

 The periplasm contains proteins distinct from those in the cytoplasm.

 Periplasmic proteins have various functions in cellular processes including:
transport, degradation, and motility.

The periplasm [86] is found in gram-negative bacteria and is the space in between the
cytoplasmic and outer membranes. (Many feel a periplasm is also present in gram-
positive bacteria in between the cytoplasmic membrane and the peptidoglycan.) The
periplasm is filled with water and proteins and is therefore somewhat reminiscent of the
cytoplasm. However, pools of small molecules in the periplasm are not like those in the
cytoplasm because the membrane prevents the free exchange between these two
compartments. Also, the proteins found in the periplasm are distinct from those in the
cytoplasm and are specifically guided to this site during translation through specific
signal sequences typically near their N-termini. Figure 2-64 [87] lists some examples of
these proteins.

The peptidoglycan shell that provides the strength to prokaryotic membranes is also
found in the periplasmic space of gram-negative bacteria, while in gram-positive
bacteria it provides the outside border to the periplasm.

Figure 2-64 Different types of periplasmic enzymes and their role in the
cell

Enzyme Type Examples Function

Hydrolytic Degrading phosphate-containing
phophatases
enzymes compounds.
proteases Degrading proteins and peptides.
endonucleases Degrading nucleic acids.
sugars, amino acids, Binding substrates and docking with
Binding proteins
norganic ions, vitamins transport protein in membrane.
Sensing the environment and changing
Chemoreceptors Chemotaxis, ermination
cell behavior in response.
Detoxifying β-lactamase Degrading penicillin and related
enzymes compounds before they get into the cell.

Periplasmic enzymes have several main functions, detecting nutrients in the

environment, degradation of polymers, and protection from harmful compounds.

2 - 25 The cell wall surrounds and holds in the microbe

 The cell wall in bacteria contains peptidoglycan, a polymer of N-acetyl

glucosamine, N-acetyl muramic acid and amino acids.
 Gram-positive cell walls contain a thick layer of peptidoglycan that encircles the
cell.
 Gram-negative cell walls contain a thin layer of peptidoglycan between the
cytoplasmic membrane and the outer membrane.

This section will restrict itself to the bacterial cell wall, but at the end of the chapter we
will compare this to archaeal cell walls. The cell wall is essential to the survival of most
microorganisms. Many microbes live in environments in relatively dilute environments
and the cell wall's most important function is to prevent the cell from bursting due to the
osmotic stress placed upon it as discussed previously in section 2-13. The cell wall also
determines the shape of the cell. Any cell that has lost its cell wall, either artificially or
naturally, becomes roughly spherical and lyses due to osmotic pressure, unless placed in
certain concentrated solutions. Finally, the cell wall helps to support any structure that
penetrates from the cell out into the environment.

Figure 2-36 A gram-positive bacterium

A Gram stain of the gram-positive bacterium Bacillus cereus

The structure and synthesis of prokaryotic cell walls is unique and many compounds
found in the bacterial cell wall are found nowhere else in nature. It is true that plants
also make cell walls, but they are chemically and structurally different. There are two
basic types of bacterial cell wall structures that have been studied in detail: gram-
positive and gram-negative. These two classes of bacterial cells look very different
following staining with the Gram stain and this has been a standard basis for starting to
identify different bacterial species. Figures 2-36 and 2-37 show Gram stains of gram-
positive and gram-negative bacteria, respectively.

Figure 2-37 A gram-negative bacterium

A Gram stain of the gram-negative bacterium Serratia marcescens.

When the Gram stain was developed by Hans Christian Gram in 1884 the molecular
basis of the stain was unknown. In fact very little was understood about bacteria in
general. He just determined empirically that when bacterial smears were run through a
four-step staining procedure using two different dyes, some cells retained the first dye
and stained purple, while other only retained the second dye and stained pink. Years
later it was discovered that the basis for this differential reaction relates to the cell wall
as shown in Figure 2-38 [88].

Figure 2-38 A comparison of the ultrastructure of gram-positive and

gram-negative cells
The different Gram reactions occur because of structural differences between the
bacterial cell walls. Gram-positive cells (Group B streptococci) appear smooth in a
scanning electron micrograph (A) and are composed of a single layer of peptidoglycan
(B). Gram-negative cells (E. coli) have an undulating surface and have three layers (C
and D). (Sources: S. H. Pincus, et al. 1992. J. Bacteriol 174:3739-3749 [panels A and
B]; M. E. Bayer and C. C. Remsen. 1970. J. Bacteriol. 101:304-313 [panel C]; T. J.
Beveridge. 1999. J. Bacteriol. 181:4725-4733 [panel D])

As shown in Figure 2-38 [89], the gram-negative cell has an additional layer and the
outside of the cell appears convoluted when compared to the gram-positive cell. The
gram-positive wall is much thicker than is the gram-negative wall and its external
appearance is smoother. gram-positive and gram-negative cells do share one thing in
common that is unique to bacteria - peptidoglycan. We will talk about the structure of
this and then move on to examine the various structures found in each cell wall type.

Peptidoglycan is a thick rigid layer composed of an overlapping lattice of two sugars,

N-acetyl glucosamine (NAG) and N-acetyl muramic acid (NAM), that are cross-linked
by amino acid bridges as shown in Figure 2-39 [90]. The exact molecular makeup of
these cross-bridges is species-specific. NAM is only found in the cell walls of bacteria
and nowhere else. Attached to NAM is a side chain generally composed of four amino
acids. In the best-studied bacterial cell walls (E. coli) the cross-bridge is most
commonly composed of L-alanine, D-alanine, D-glutamic acid and diaminopimelic acid
(DPA).

Figure 2-39 The chemical structure of peptidoglycan

The generalized peptidoglycan monomer showing the two sugars that make up the
backbone. The R group consists of four amino acids, with the best-studied cell walls
containing L-alanine, D-alanine, D-glutamic acid and diaminopimelic acid.

Note that peptidoglycan contains D-amino acids, which are different than the L-amino
acids found in proteins. D-amino acids have the identical composition as L-amino acids,
but are their mirror images. The use of D-amino acids is unusual in biology and bacteria
have enzymes called racemases to convert between D and L forms specifically for this
use.

The NAM, NAG and amino acid side chain form a single peptidoglycan unit that can
link with other units via covalent bonds to form a repeating polymer. The polymer is
further strengthened by covalent bonds between cross-bridges and the degree of cross-
linking determines the degree of rigidity. In the E. coli, the penultimate D-alanine of
one unit is linked to DPA of the next cross-bridge. In some gram-positive microbes
there is a peptide composed of various amino acids that serves as a link between the
cross-bridges. For example, in Staphylococcus aureus strains, five glycines make up the
linker between peptidoglycan monomers. The sequence of these linkers varies
considerably between species. The completed peptidoglycan layer forms a strong mesh
that can be thought of as a chain link fence. The complete cell wall contains one or more
layers of peptidoglycan one atop the other, providing much of the strength of the cell
wall.

While both gram-negative and gram-positive bacteria have peptidoglycan, its physical
arrangement in the cell wall is different. In gram-positive cells the peptidoglycan is a
heavily cross-linked woven structure that encircles the cell in many layers. It is very
thick with peptidoglycan accounting for 50% of weight of cell and 90% of the weight of
the cell wall. Electron micrographs show the peptidoglycan to be 20-80 µm thick. In
gram-negative bacteria the peptidoglycan is much thinner with only 15-20% of the cell
wall being peptidoglycan and it is only intermittently cross-linked. In both cases
peptidoglycan is not a barrier to solutes, as the openings in the mesh are large enough
for most molecules including proteins to pass through. Figure 2-40 [91] shows an artist's
rendering of what the structure might look like.

Figure 2-40 A cartoon of the peptiodglycan mesh

The peptidoglycan polymers then crosslink with other peptidoglycan chains to form a
complex mesh that wraps the cell in a structure a kin to chicken wire.

There are numerous antibacterial agents that target the bacterial cell wall because
mammals do not synthesize walls and therefore are not susceptible to the toxic effects of
these agents. Penicillin inhibits the linking of the amino acid side chains of
peptidoglycan units, which therefore weakens the stability of the wall eventually,
causing the cells to rupture. Humans and other animals even synthesize an enzyme that
specifically attacks bacterial cell walls. The enzyme lysozyme is found in many body
fluids and hydrolyzes the NAM-NAG bond in the cell wall. It serves as a critical part of
the mammalian defense against bacterial invasion. Figure 2-41 [92] shows a depiction
of the gram-positive cell wall.

Figure 2-41 The gram-positive cell wall

The cell wall is made mostly of peptidoglycan, interspersed with teichoic acid which
knits the different layers together. The amount of crosslinking is higher and the wall is
thicker than in gram-negative cell walls.

The gram-positive cell wall

Another structure in the gram-positive cell wall is teichoic acid. It is a phosphodiester

polymer of glycerol or ribitol joined by phosphate groups. Amino acids such as D-
alanine are attached. Teichoic acid is covalently linked to muramic acid and stitches
various layers of the peptidoglycan mesh together. Teichoic acid stabilizes the cell wall
and makes it stronger. The chemical formula of teichoic acid is shown in Figure 2-42
[93]

Figure 2-42 Teichoic acid

Teichoic acid is a long, thin molecule that weaves through the peptidoglycan.

Gram-negative cell structure

Gram-negative cell walls have a more complicated structure than do those of gram-
positive organisms. Outside the cytoplasmic membrane is the periplasm, which contains
the thin layer of peptidoglycan. The peptidoglycan in gram-negative cells contains less
cross-linking than in gram-positive cells with no peptide linker. Covalently bound to the
peptidoglycan is Braun's lipoprotein, which has a hydrophobic anchor in the outer
membrane that helps to strongly bind the peptidoglycan to the outer membrane. Figure
2-43 [94] shows the arrangement of the gram-negative cell wall.

Figure 2-43 The gram-negative cell wall

The cell wall in gram-negative bacteria contains much less peptidoglycan and is
surrounded by an outer membrane. There is much less crosslinking between the
peptidoglycan. LPS is also present in the outer membrane and penetrates into the
surrounding environment.

The outer membrane

The outer membrane of gram-negative bacteria is another lipid bilayer similar to the
cytoplasmic membrane, and contains lipids, proteins, and also lipopolysaccharides
(LPS). The membrane has distinctive sides, with the side that faces the outside
containing all the LPS. LPS is composed of two parts: Lipid A and the polysaccharide
chain that reaches out into the environment. Lipid A is a derivative of two NAG units
with up to 7 hydrophobic fatty acids connected to it that anchor the LPS in the
membrane as shown in Figure 2-44 [95]. Attached to Lipid A is a conserved core
polysaccharide that contains KDO, heptose, glucose and glucosamine sugars. The rest
of the polysaccharide consists of repeating sugar units and this is called the O-antigen.
The O-antigen varies among bacterial species and even among various isolates of the
same species. Many bacterial pathogens vary the make-up of the O-antigen in an effort
to avoid recognition by the host's immune system.

Figure 2-44 The structure of LPS

LPS is composed of three sections: the lipid A region, a conserved core polysaccharide,
and a highly variable O-polysaccharide. (A) The chemical structure of LPS. (B) A
molecular model of the membrane from Pseudomonas aeruginosa. Source (T. P.
Straatsma, Pacific Northwest National Laboratory)

LPS confers a negative charge and also repels hydrophobic compounds including
certain drugs and disinfectants that would otherwise kill the cell. Some gram-negative
species live in the gut of mammals and LPS repels fat-solubilizing molecules such as
bile that the gal bladder secretes. This repulsion enables these bacteria to survive in this
environment. The O-antigen and other molecules on the outer membrane are also used
by certain viruses that infect bacteria, as a means to identify the correct hosts for
infection.

LPS is medically important because when LPS is released from bacterial cells it is toxic
to mammals and is therefore called endotoxin [96]. It creates a wide spectrum of
physiological reactions including the induction of a fever (endotoxins are said to be
pyrogenic [97]), changes in white blood cell counts, leakage from blood vessels, tumor
necrosis and lowered blood pressure leading to vascular collapse and eventually shock.
At high enough concentrations the LPS endotoxin is lethal. Finally, the outer membrane
keeps the enzymes in the periplasm from floating away from the cell.
There are fewer total proteins and fewer unique types of proteins in the outer membrane
than in the cytoplasmic membrane. Porins are particularly important components
because of their role in the permeability of the outer membrane to small molecules.
Porins are proteins that form pores in the outer membrane large enough to allow passage
of most small hydrophilic molecules. Figure 2-45 [98] shows the structure of a porin at
the molecular scale. All known porins have a similar structure, with the protein
containing a central channel that allows the passage of molecules. This allows migration
of these molecules into the periplasmic space for possible transport across the
cytoplasmic membrane. Some porins in the outer membrane are general, doing simple
discrimination on size and charge, but having little substrate specificity. Examples
include OmpF that is selective for positively charged molecules and PhoE that is
permeable to negatively charged molecules. Other porins are more specific. The best
studied is LamB, which recognizes the sugar polymer maltooligosaccharide and
transports it through the outer membrane. Very large or hydrophobic molecules cannot
penetrate the outer membrane, so the outer membrane serves as a permeability barrier to
at least some molecules.

Figure 2-45 The structure of a porin

The molecular structure of a porin. The view in (A) is from the outside of the cell
looking at the membrane surface. The view in (B) is the perspective from the side (i.e.
from the membrane). The porin has three protein subunits and the actual pore is the
central triangular area in the top panel formed by the three subunits.

There are also other types of outer membrane proteins that are involved in various
functions. OmpA in E. coli seems to connect the outer membrane to the peptidoglycan.
Some pathogens contain outer membrane proteins that help them neutralize host
defenses. Finally all gram-negative bacteria contain high molecular weight proteins
involved in the uptake of large substrates such as iron-complexes and vitamin B12.

The differences between the cell walls of gram-positive and gram-negative bacteria
greatly influence the success of the microbes in their environments. The thick cell wall
of gram-positive cells allows them to do better in dry conditions because it reduces
water loss. The outer membrane and its LPS helps gram-negative cells excel in the
intestines and other host environments. Figure 2-65 [99] summarizes the difference
between gram-negative and gram-positive cell walls.
Figure 2-65 Properties of cell walls

Property gram-positive gram-negative

Thickness of wall 20-80 nm 10 nm
Number of layers in wall 1 2
Peptidoglycan content >50% 10-20%
Teichoic acid in wall + -
Lipid and lipoprotein content 0-3% 58%
Protein content 0% 9%
Lipopolysaccharide 0 13%
Sensitive to penicillin Yes Less sensitive
Digested by lysozyme Yes Weakly

A summary of the differences between gram-positive and gram-negative cell walls.

2 - 26 Some bacteria lack cell walls

For most bacterial cells, the cell wall is critical to cell survival, yet there are some
bacteria that do not have cell walls. Mycoplasma species are widespread examples and
some can be intracellular pathogens that grow inside their hosts. Cell walls are
unnecessary here because the cells only live in the controlled osmotic environment of
other cells. It is likely they had the ability to form a cell wall at some point in the past,
but as their lifestyle became one of existence inside other cells, they lost the ability to
form walls. Consistent with this very limited lifestyle within other cells, these microbes
also have very small genomes. They have no need for the genes for all sorts of
biosynthetic enzymes, as they can steal the final components of these pathways from the
host. Similarly, they have no need for genes encoding many different pathways for
various carbon, nitrogen and energy sources, since their intracellular environment is
completely predictable. Because of the absence of cell walls, Mycoplasma have a
spherical shape and are quickly killed if placed in an environment with very high or
very low salt concentrations. However, Mycoplasma do have unusually tough
membranes that are more resistant to rupture than other bacteria since this cellular
membrane has to contend with the host cell factors. The presence of sterols in the
membrane contributes to their durability by helping to increase the forces that hold the
membrane together.

Other bacterial species occasionally mutate or respond to extreme nutritional conditions

by forming cells lacking walls, termed L-forms. This phenomenon is observed in both
gram-positive and gram-negative species. L-forms have varied shapes and are sensitive
to osmotic shock.

2 - 27 The cell surface extends into the environment

Surface structures are typically attached to a membrane, and extend into the
environment. Important structures include flagella [100], pili [101], fimbriae [102], and
glycocalyx [103]. These protrusions and surfaces interact with the environment around
the microorganism and therefore are pivotal in how the microbe sees the world and how
we see the microbe.
2 - 28 Flagella are one type of structure used for motility

 Flagella are semi-rigid structures used to move microbial cells.

 Flagella are mostly composed of flagellin, a protein polymer of repeated
subunits. Flagella are attached to the cell through a complex of proteins called
the hook and basal body.
 Flagella cause the cell to move by their rotation, which is powered by the proton
motive force.

Surface structures are typically attached to a membrane and extend into the
environment. Important structures include flagella [104], pili [105], fimbriae [106], and
glycocalyx [107]. These protrusions and surfaces interact with the environment around
the microorganism and therefore are pivotal in how the microbe sees the world and how
we see the microbe.

In many bacteria, flagella are responsible for motility in liquid. There is also a loose
correlation between cell shape and the presence of flagella. Almost all spirilla, half of
all rod-shaped bacteria, and only a few of the cocci are motile by flagella. In fact, most
cocci are non-motile. One rationale for this correlation might be that spherical cells such
as the cocci simply do not have the best geometry for directional movement by flagella,
unlike more linear bacteria.

Flagella can be thought of as little semi-rigid propellers that are free at one end and
attached to a cell at the other. Flagella are thin (20 µm) and long with some having a
length 2-3 times (about 10 µm) the length of the cell. Due to their small diameter,
flagella cannot be seen in the light microscope unless a special stain is applied. Bacteria
can have one or more flagella arranged in clumps or spread over the cell surface. Figure
2-46 demonstrates some of the more common arrangements.

Figure 2-46 Flagellar arrangements

A cartoon of several common flagellar arrangements.

Chemical Structure

Flagella are mostly composed of the protein flagellin, which is bound in long chains and
wraps around itself in a left-handed helix as shown in Figure 2-47 [108]. The number of
protein monomers that it takes to make a single turn of the helix is determined by the
protein subunits themselves.

Figure 2-47 Flagella attachment in bacteria

Flagella are attached by a hook and rings that anchor it to the cell wall of the
microorganism. In gram-positive bacteria (A) the rings are located in the cytoplasmic
membrane and the flagella passes through the peptidoglycan to the outside environment.
In gram-negative bacteria (B) there are additional protein rings in the outer membrane.

The flagellum is attached to the cell through complex protein structures termed the hook
and the basal body. One ring in the basal body rotates relative to the other causing the
flagellum to rotate. The energy to drive the basal body is obtained from the proton
motive force. In some fashion the translocation of protons from outside to inside the
membrane causes the rotation of the flagellum. In a sense, the protons move through the
wheel-like structure of the basal body (similar to a water wheel, except using protons)
and this causes the rotation of the assembly including the flagellum. When E. coli is
swimming through a solution the flagella turn counter-clockwise and push the microbe
through solution. This behavior is termed smooth swimming. It is possible for E. coli to
also reverse the direction of flagellar rotation and when the flagella turn clockwise, they
pull against the bacterial cell. Since E. coli is flagellated peritrichously (that is, at many
positions), it is pulled in all directions and tumbles.

How fast do bacterial cells move? They average 50 µm/sec, which is about 0.00015
kilometers/hr. This may seems slow but remember their tiny size. Figure 2-66 [109]
shows a better comparison and indicates that relatively speaking, bacteria are faster than
humans. Also remember that this motility happens in water, which is much more
viscous than air.

Figure 2-66 Relative speeds of organisms

Organism Kilometers per hour Body lengths per second

Cheetah 111 25
Human 37.5 5.4
Bacteria 0.00015 10

Bacteria seem slow, until you consider relative size. Then they are quite fast.
If a flagellum breaks off it is resynthesized until it reaches the appropriate length. This
growth actually occurs from the tip. The flagellar filament is hollow and flagellin
monomers are passed up through this space until they reach the growing tip and are
added to the structure.

2 - 29 Advantages of motility

 Motility allows microbes to move toward desirable environments and away from
undesirable ones. Some common stimuli include chemicals, light and oxygen.

Typically microbes in aqueous environments continually move around looking for

nutrients. Even microorganisms in the soil have uses and opportunities for movement.
Sometimes this movement is random, but in other cases it is directed toward or away
from something. As a rough guide, bacteria want to move towards food or energy
sources and away from toxic compounds. In other words, bacteria are capable of
showing simple behavior that depends upon various stimuli.

Directed Motility

There are several classifications of tactic responses and the category is based upon the
stimulus that the movement is responding to.

 chemotaxis - towards or away from a chemical stimulus

 phototaxis - towards light
 aerotaxis - towards or away from oxygen

The purpose of chemotaxis should be fairly evident: attraction to nutrients or avoidance

of damaging compounds. A cell distinguishes chemicals that fall into either of these
classes with receptor molecules near their surfaces that can tell if specific chemicals are
in the environment. Many of these receptors are also involved in transport of their target
molecules. Now it actually is trickier than it sounds. A cell does not merely want to
sense the presence of nutrient, but wants to move toward the highest concentration of it.
It does this by an amazing process that is sort of a primitive memory, where it
essentially asks if its receptors are binding more or less of the compound than they were
a moment ago ("a moment ago" being about 200 milliseconds.). If they are binding
more, the cell keeps swimming, but if they are binding less, it tumbles and then swims
in a random direction. This tumbling is the direct result of E. coli's ability to reverse the
rotation of its flagella.

Phototaxis is somewhat different, but again the cells move toward optimal levels of
specific wavelengths of light. Rhodobacter sphaeroides (a photosynthetic microbe)
performs phototaxis by a mechanism analogous to chemotaxis in E. coli, but there are
important differences. R. sphaeroides contains only one polar flagella, which rotates
during a run toward light. If, during a run, light conditions worsen, the flagella stop
rotating (instead of reversing its rotation). The microbe instead depends upon Brownian
movement in the environment to turn it in a new direction. R. sphaeroides is also
capable of modulating the speed of rotation of the flagella in response to the
environment, swimming faster when conditions are improving and slower if conditions
are deteriorating.
Aerotaxis refers to the ability of some bacteria to be respond to the presence of oxygen.
It is mechanistically generally similar to what has just been described and depends on
levels of dissolved oxygen in the environment, but whether a bacterium swims toward it
or away depends on the type of metabolism that it has.

2 - 30 Some bacteria move by gliding motility

 Gliding motility is an energy-requiring process that allows bacteria to move over

a solid surface.
 Several different groups of microbes are capable of gliding motility including
Myxococcus, Neisseria, Pseudomonas, Cytophaga, and Flavobacterium.
 There may actually be several mechanisms of gliding motility. One mechanism
involves Type IV pili, using it as a tether. A second method involves extrusion
of an extracellular polysaccharide. A final proposed mechanism may involve
rotation of basal-body-like structure on the surface of the microbe.

Flagella are not the only means bacteria have developed for moving about the
environment. A large collection of phylogenetically diverse bacteria have developed
gliding motility [110], which is an energy-requiring process by which bacteria move
smoothly over a solid surface. Gliding microbes tend to be predominately gram-
negative, but there are examples of gram-positive bacteria. This type of motility is not
as well understood as flagellar propulsion, and there appear to actually be several
different mechanisms employed to accomplish it.

In one mechanism the microbes use pili (type iv pili [111] specifically) that are extended
away from the cell and stick to the surrounding surface. The microbe then pulls itself
toward the tethered end by retracting the pilus back inside the cell. By repeating this
process the cell drags itself along a surface. This type of motility is observed in a
number of microorganisms including Pseudomonas aeruginosa, Neisseria gonorrhoeae,
and Myxococcus xanthus. Movement in this manner requires energy in the form of ATP.

There are also gliding bacteria that use other mechanisms. Some filamentous
cyanobacteria seem to extrude an extracellular polysaccharide through small pores on
the surface of the organism. Figure 2-48 [112] depicts this type of gliding motility. It is
thought that the polysaccharide exiting from the cell propels the microbe along the
surface by some means.

Figure 2-48 Gliding motility in Paenibacillus

Colonies of vegetative cells of a Paenibacillus isolate moving across an agar surface

using gliding motility. (Source: Jon Roll, University of Wisconsin-Madison)

A third groups of microbes (Cytophaga and Flavobacterium) seem to use yet another
mechanism that is dependent upon proton motive force. Observation of these microbes
in the presence of very small latex beads show the beads moving along the surface of
the microbe in a directional manner. The bead moves from one pole of the rod-shaped
bacteria to the other, sometimes reversing direction. One model to explain this behavior
is the concerted movement of cytoplasmic membrane proteins that are coupled to outer
membrane proteins. These membrane proteins might form a kind of conveyor belt. The
outer membrane proteins in this mechanism are in contact with the environmental
surface and their movement propels the microbe forward.

Interestingly, Myxococcus xanthus uses several of these mechanisms in its motility. M.

xanthus is a social predator. It glides around in large groups of cells secreting toxins and
degradative enzymes that kill other microbes. The leftovers of these dead microbes then
provide nutrients for the marauding Myxococcus. Investigations by Dale Kaiser and
others have revealed that this microbe actually has two types of gliding motility, social
and adventurous. Social motility occurs in groups while adventurous motility involves
single cells. It turns out that social motility is dependent upon pili as described above,
while adventurous motility involves the extrusion of slime from pores in the microbe's
cell wall. Dr. Kaiser has a collection of interesting images and movies [113] that
describe this interesting microbe

2 - 31 Pili and fimbriae are involved in adhesion, motility and DNA

exchange

 Pili and fimbriae are smaller than flagella, but have a similar structure
 Pili can serve in DNA exchange.
 Pili and fimbriae are often involved in attachment to surfaces and are important
for biofilm formation.

Pili and fimbriae are structurally similar to flagella and are composed of one or a few
proteins arranged in a helical fashion. Figure 2-49 [114] shows pili isolated from
Neisseria gonorrhea. Each protein subunit assembles on the growing structure at the tip,
as is the case with flagella. There are a number of genes necessary for the successful
construction of pili and their products might perform functions such as moving the
structural proteins across the membrane, methylating the structural proteins or retracting
the pilus. The same is generally true for fimbriae.

Figure 2-49 Pili and fimbriae

The pili shown in this micrograph are those of Neisseria gonorrhea with Tobacco
Mosaic virus (the thicker structures) added as a size reference. The length of the
Tobacco Mosaic virus is 0.05 µm. (Source: Katrina Forest, University of Wisconsin-
Madison)

Fimbriae are found on many bacteria and are shorter and straighter than flagella and are
more numerous. Not all bacteria synthesize them. Fimbriae do not function in motility,
but are thought to be important in attachment to surfaces. Some microbes attach to hosts
by fimbriae, and successful colonization of many surfaces is totally dependent upon the
ability to make fimbriae. Swarming microbes such as Myxococcus use them to sense the
presence of similar microbes, which helps keep their "hunting packs" together.

Pili are longer than fimbriae and there are only a few per cell. They are known to be
receptors for certain bacterial viruses, but certainly the bacterium makes them for
another purpose. There are two basic functions for pili: gene transfer and attachment to
surfaces. In genetic transfer in a broad variety of bacteria, a donor bacterium attaches to
a recipient by the sex pilus. Then the donor cell depolymerizes the pilus at the end that
is attached to itself, which draws the cells together and eventually a small pore is
created between the two cells. DNA is then transferred through that pore from the donor
to the recipient and the cells separate. For a long time, it was thought that the donor
bacterium's genome passed through the sex pilus into the recipient, but this is certainly
not the case. Transfer of genes this way is not restricted to related species, which
implies that a pilus from one organism can attach to a variety of others. Conjugation, as
this transfer process is known, is one explanation for the rapid spread of drug resistance
in many different species of bacteria and is covered in depth in the Chapter on Genetics
and Genomics.

Pili have also been show to be important for the attachment of many types of
microorganisms to surfaces. For example, Neisseria gonorrhoeae, the causative agent of
gonorrhea, has a special pilus that helps it adhere to the urogenital tract of its host. The
microbe is much more virulent when able to synthesize pili.

2 - 32 Bacterial cells are often covered in glycocalyx

 The outer surface of cells is often covered in a polysaccharide or protein

material called glycocalyx.
 Glycocalyx can be formed from protein, polysaccharides, polyalcohols or amino
sugars.
 Glycocalyx can function in attachment, protection from desiccation, and
protection from attack by a hosts immune system.

The general term for any network of polysaccharide or protein containing material
extending outside of the cell is the glycocalyx [115]. Many bacteria produce such a
coating on the outside of their cell, and they come in two types: capsules and slime
layers. The difference between the two is somewhat arbitrary. A capsule is closely
associated with cells and does not wash off easily, while a slime layer is more diffuse
and is easily washed away. Figure 2-50 [116] shows the capsule surrounding Klebsiella
planticola.

Figure 2-50 Capsule surrounding cells of Klebsiella planticola

The capsule (made of polysaccharide) in the figure is colorless and about the diameter
of the cell. The background is darker.

There are many different types of proteins, polysaccharides, polyalcohols and amino
sugars in glycocalyx and the exact makeup is species-specific. The structure can be
thick or thin, rigid or flexible. Observing cells stained with India ink in the microscope
shows dark cells with an outline around them, as the stain does not penetrate the
glycocalyx.

There are several functions attributed to glycocalyx, one of which is to help cells attach
to their target's environment. Streptococcus mutans produces a slime layer in the
presence of sucrose. This forms a surface that allows other bacteria to aggregate on
tooth surfaces and results in dental plaque. Vibrio cholerae, the cause of cholera, also
produces a glycocalyx that helps it attach to the intestinal villi [117] of the host.
Glycocalyx can play other roles in pathogenesis as well. Bacteria that enter the body are
always in danger of being attacked by phagocytes (host cells that protect you from
invaders). Often the capsule makes it more difficult for phagocytes [118] to attach to and
engulf pathogens. As one example, Streptococcus pneumoniae, when encapsulated by a
glycocalyx, is able to kill 90% of infected animals, while non-encapsulated forms
cannot kill. In addition capsules and slime layers are largely hydrophilic, so they can
bind extra water in the environment and contribute to the protection of the cell from
desiccation. Capsules and slime layers can also provide protection from the loss of
nutrients by holding them within the layer. These extra layers coating the surface of the
cell may also potentially mask viral receptors making it more difficult for viruses to
attach. Many of these functions manifest themselves in the form of biofilms, which
allow the formation of communities of microorganisms all held together by glycocalyx.
Biofilms are covered in more detail in the chapter on Environmental Microbiology.

2 - 33 Bacteria can exist in different cell states

 Some microbes are capable of forming resting structures that allow them to
survive harsh conditions. When conditions become more favorable, they
germinate and continue to multiply.
 Spores are formed by a wide variety of microbes and have very low to
nonexistent rates of metabolism.

For obvious reasons we have focused on growing cells, but there are non-growing states
of microbes that are important to both microbes and humans. In these states, termed
spores and cysts, the cells remain dormant for long periods of time. Part of the relevance
of these states is that the very properties that allow the cell to survive extended time
periods also happen to make the cells resistant to our typical efforts to kill them. As a
consequence, the attempt to sterilize a sample can be thwarted by the presence of
bacterial spores or cysts. In this section we examine some of the properties of these
structures.

Spores and cysts

Spores and cysts are resting structures. That is, these states have very low to nonexistent
rates of metabolism. They are common in organisms that live in soil and may need to
survive some rough conditions such as lack of nutrients, high heat, radiation, or drying.

Sporulation is a unique developmental cycle. After the decision to sporulate is made,

creation of a different type of cell needs to take place, which requires turning on a large
collection of genes in a tightly coordinated fashion. In addition, all of this expression
has to be finished before the microbe runs out of energy. There are several types of
spores. Some are highly resistant structures that are formed under conditions of cell
stress and are created inside a supportive cell and are termed endospores [119]. Others
are part of the normal reproductive cycle, being created by differentiation of a
vegetative cell and we will refer to these as spores. In this section we talk generally
about the structure of spores and in the chapter on Regulation we will examine the
regulation of sporulation.

2 - 34 Endospores are very resistant structures

 Endospores are a subclass of spores that are very resistant to harsh conditions.
They can survive high heat (>100 °C), drying, radiation, and many damaging
chemicals.
 Endospores can germinate many years after formation, even millions of years
later.
 Endospores contain four layers, core, cortex, coat, and exosporium.

Endospores are refractile - light cannot penetrate them - so that they are very easy to see
in the phase microscope and this makes it easy to detect them. Most endospores [120]
have no measurable metabolism [121] and are really a form of suspended animation.
Endospores can survive for a very long time, and then return to a growing state, a
process termed germination. Endospores that were dormant for thousands of years in the
great tombs of the Egyptian Pharaohs were able to germinate and grow when placed in
appropriate medium [122]. Several scientists have been able to recover viable
endospores from bees trapped in amber that is 25-40 million years old. The microbe
isolated was found to be most closely related to Bacillus sphaericus. There are even
claims of endospores that are over 250 million years old being able to germinate when
placed in appropriate medium, but these claims still need to be verified. Endospores are
found everywhere, are easily dispersed throughout the environment and can be difficult
to remove. The anthrax scare of 2001 in the United States is ample evidence of the
insidiousness of endospores and their impressive resistance.

Endospores are resistant to heat (>100 °C), radiation, many chemicals (i.e. acids, bases,
alcohol, chloroform), and desiccation. The mechanisms that account for this resistance
include the impermeability of the endospore coat, the dehydration of the cytoplasm [123]
and the production of special proteins that protect the spores DNA [124]. Figure 2-51
[125] shows the major structures of an endospore.

Figure 2-51 An endospore

An electron micrograph of an endospore of Bacillus subtilis showing the core, cortex
and coat. (Source: M. Serrano, et al. 1999. J. Bacteriol. 181:3632-3643)

The formation of an endospore is clearly a great advantage for these bacteria [126] and
enables them to endure extreme stress. At a later time, even much later, when conditions
are favorable, they can reemerge and flourish. Endospores enable a species [127] to
spread easily from one suitable environment to another an many endospore-forming
bacteria are ubiquitous in the environment. Endospores are a particular problem in the
food industry where great care must be taken to insure either the destruction of
endospores or suitable preservation methods so that endospore-forming bacteria (and
other microbes) cannot grow.

Endospores can be divided into several important parts (Fig 2-51). The center of the
endospores contains the core and it consists of the cytoplasm, DNA, ribosomes,
enzymes and everything that is needed to function once returned to the vegetative state.
The core is dehydrated, which is essential for heat resistance, long-term dormancy and
full chemical resistance. Calcium dipicolinate is a major component of the core and has
been shown to play a role in resistance to wet heat and UV [128] light. The cortex
surrounds the core and is composed of two layers, a thin more densely staining layer
that is similar in structure to the vegetative cell [129] wall and a thicker less dense layer
containing modified peptidoglycan. Two major modifications are present. First, there is
less cross-linking with only 3% of the muramic acid present in the peptidoglycan of the
cortex participating, in comparison to 40% of muramic acid in the vegetative cell wall.
Second, much of the muramic acid is modified to a muramic-β-lactam structure. Both of
these modifications of the cortex appear to be important in germination. Muramic-β-
lactam serves as a specific target for lytic enzymes that are activated during germination
and the lower cross-linking enables easier outgrowth. Outside of the cortex is the spore
coat [130] containing several protein [131] layers that are impermeable to most
chemicals. The coat is composed of more than two dozen different types of proteins and
there is some evidence that these proteins are connected by cross-links. This covalent
connection between coat proteins probably contributes to the spores' resistance.

2 - 35 Some microbes make other types of spores

 Less resistant resting structures (spores and cysts) are formed by other microbial
species.
 Spores and cysts are less resistant to harsh treatments than are endospores, but
do have some resistance especially to desiccation.

Endospores are not the only type of spore made by microorganisms. Azotobacter
species and several others are know to form cysts, which are dormant cells with
thickened cells walls. Cysts are resistant to desiccation and some chemicals, but cannot
withstand high temperatures as endospores can. The actinomycetes are a large group of
spore-forming, gram-positive bacteria that grow by forming long tubules called
filaments. Under nutrient poor conditions these filaments differentiate into round resting
structures termed spores. In contrast to endospores, these structures are part of the
reproductive process. The developmental process to create an actinomycete spore is less
complex than that of the endospore. It involves the simple formation of cross walls that
divide the filament into sections, each containing a chromosome. These then
differentiate into mature spores. During this process a tougher cell wall is laid down and
there is conversion of the cytoplasm to a dormant state so that the spore becomes more
resistant to heat and chemicals, though not as hardy as an endospore. Actinomycete
spores are capable of surviving for long periods of time (for years) and can germinate
into vegetative cells when appropriate growth conditions are present. Many different
genera are capable of forming this type of spore and the ability to form these structures
does not seem to correlate with any group of microorganisms.

Figure 2-52 A cyst

The micrograph shows cysts and cells of Azotobacter vinelandii at 1000 X

magnification. The cysts are the phase-bright objects, while the cells are darker.

2 - 36 Heterocysts are differentiated cells that specialize in nitrogen

fixation

 Some cyanobacteria species form specialized structures for the fixation of

nitrogen.

Some photosynthetic filamentous cyanobacteria are capable of forming specialized

structures called heterocysts [132]. These are rounded structures distributed at regular
intervals along the string of vegetative cells as shown in Figure 2-53 [133] or at one end.
Heterocysts evolved to solve the problem of performing plant-like photosynthesis
(which produces oxygen) and at the same time fixing N2 to ammonia (a process that
involves enzymes that are inactivated by O2). The single focus of the heterocysts is to
fix N2, while the rest of the cells perform photosynthesis (and divide), thus keeping the
two processes separate. Heterocysts develop a surface that is impermeable to gasses,
and begin synthesizing large amounts of nitrogenase, the protein that fixes N2.
Importantly, these heterocysts maintain some permeability to the cells on either side.
Neighboring cells take N2 from the atmosphere and pass it along to the heterocyst,
which reduces it to NH3 and returns fixed nitrogen to its neighbors. The neighboring
cells also take up and utilize O2, but they prevent it from reaching the heterocyst.
Heterocysts are essentially specialized organs for the "multi-cellular organism"
represented by a chain of cyanobacterial cells and they are only formed when nitrogen is
limiting. The regulation of this developmental cycle is intriguing and serves as a simple
example of multicellular development in a unicellular organism.

Figure 2-53 The heterocyst of the filamentous cyanobacteria Anabaena

Heterocysts are common in several different groups of cyanobacteria and are the site of
nitrogen fixation. Note the slightly enlarged size and distinct shape of the heterocyst
when compared to the vegetative cells on either side. (Source: Michael Clayton,
University of Wisconsin-Madison)

This ends our survey of the cellular structure of bacteria. In the remaining sections we
take a look at the major structural differences that distinguish the archaea and
eukaryotes from the bacteria.

2 - 37 Archaea, a different type of microbe

 Archaea refers a domain of organisms that was discovered in the 1970s.

 They are a unique form of life, but have commonalities with both bacteria and
eukaryotes.

Their discovery

Before 1977 prokaryotes were phylogenetically organized into one group termed the
Monera. The discovery of the extreme thermophile Thermus aquaticus in the hot
springs of Yellowstone National Park by Tom Brock and the subsequent analysis by
Carl Woese began a series of experiments that would change how scientists think about
the organization of life on this planet. This revolution is the subject of later chapters, but
here we will discuss the structural differences between this new group, the Archaea,
and the Bacteria. "Bacteria" and "Archaea" refers to the formal phylogenetic
classification of organisms, but we will typically refer to them more casually as groups
of organisms and then use "bacteria [134]" and "archaea [135]". The third domain
consists of eukaryotic organisms, both microbes and multi-cellular and is referred to as
Eukarya [136]. As explained early in this chapter, it has become evident that eukaryotes
arose when certain bacteria became engulfed in archaeal cells, eventually becoming
organelles. Not surprisingly then, Archaea is a group of microbes that share some things
in common with Bacteria, others with Eukarya and have still other properties that are all
their own. In the following section we highlight some of the major structural features of
the Archaea.

Figure 2-67 Comparison of properties between Archaea, Bacteria and

Eukarya

Property Bacteria Archaea Eukarya

12 proteins (RNA
RNA 4 proteins Rifampicin 8-10 proteins Rifampicin
pol II) Rifampicin
polymerase sensitive resistant
resistant
Transcription Variable often contains
TATA box TATA box
start site a -35 and -10 region
Starting amino
formylmethionine methionine methionine
acid
Lipids ester-linked ether-linked ester-linked
pseudopeptidoglycan or
G+ peptidoglycan
Cell wall S-layer of proteins,
G- peptidoglycan and none or cellulose
composition glycoproteins, or
outer membrane
polysaccharides

Archaea are a unique form of life, as different phylogenetically from bacteria as they are
from eukaryotes.

2 - 38 The major differences between Archaea and other domains of life

 The RNA polymerase in archaea is similar to RNA polymerase II in eukaryotes.

 The ribosomes of Archaea are similar in structure to those of Bacteria.
 Lipids in membranes from Archaea are unique, containing ether linkages
between the glycerol backbone and the fatty acids, instead of ester linkages.
 The cells walls of Archaea are chemically and structurally diverse and do not
contain peptidoglycan.

RNA polymerase

DNA-dependent RNA polymerases in bacteria and archaea are different from each
other. The primary RNA polymerase of all organisms is responsible for creating
messenger RNA that is then translated into proteins at the ribosome. Bacterial RNA
polymerase is relatively simple, containing 4 different proteins. In contrast, RNA
polymerase from methanogens and halophiles (both Archaea) contains 8 proteins. In
hyperthermophiles (Archaea) RNA polymerase is even more complex, containing 10
proteins. None of the archaeal RNA polymerases are affected by the antibiotic
rifampicin, a known inhibitor of the bacterial RNA polymerase. The eukaryotic RNA
polymerase responsible for most mRNA transcription (termed RNA polymerase II)
contains 12 proteins and is similar to the RNA polymerase in the archaea.

RNA polymerases from all organisms recognize a variety of start sequences or

promoters. A promoter for mRNA transcription in bacteria is recognized by the (sigma
[137]) protein and has two recognition zones about 10 and 35 bases before the
transcription start site. The exact sequence recognized by RNA polymerase depends
upon the σ factor that has bound to the enzyme, and cells have different numbers of σ
factors that bind to different types of promoter sequences. This allows regulation of
mRNA expression by changing the levels of σ factors inside the cell. In archaea and
eukaryotes the transcription recognition sequence is a TATA sequence (termed the
TATA box [138]) and transcription is regulated by various protein transcription factors
that bind to regions near the TATA box and then recruit RNA polymerase.

Figure 2-68 Control elements in bacteria and archaea

The best-studied bacteria have promoters at two positions upstream of the transcription
start site that are recognized by the σ factors associated with RNA polymerase. The
transcriptional control elements in the archaea are much more like those found in
eukaryotes, where RNA polymerase is recruited to a promoter by interaction with
protein regulatory factors, and transcription begins downstream of a TATA box.

Translation and ribosomes

The structural make-up of the ribosomes of bacteria and archaea are similar in many
respects. The ribosomes of archaea and bacteria are of the same size (70S) and are
smaller than those of eukaryotes (80S). However, most of the ribosomal proteins,
translation factors and tRNAs of archaea more closely resemble their counterparts in
eukaryotes. In fact, mixing experiments with ribosomes from E. coli (Bacteria),
Sulfolobus (Archaea) and yeast (Eukarya) have shown functional substitution between
Archaea and Eukarya. Specifically, when the small subunit from a yeast ribosome was
mixed with the large subunit from Sulfolobus, the hybrid ribosome was capable of
translation in vitro. When the small subunit from yeast was mixed with the large subunit
of E. coli, translation did not occur. Also, in all organisms translation begins at a start
codon (typically an AUG in the mRNA). In bacteria, this codon causes the insertion of
formylmethionine [139], while in archaea and eukaryotes, it results in insertion of an
unmodified methionine.
Lipids

Archaeal membranes have features unlike those found in either eukaryotes or bacteria.
The lipids in archaea have a different chemical make-up in the following way.
Remember that lipids in bacteria are amphipathic molecules (i.e. having both
hydrophobic and hydrophilic portions) containing a backbone of glycerol connected to a
hydrophilic head group and two hydrophobic long-chain fatty acids. In eukaryotes and
bacteria the fatty acids are attached to the glycerol backbone by ester bonds, while in
archaea ether linkages are used. Also, the stereochemistry of lipids from archaea is
primarily of the S form, while that of bacteria and eukaryotes is of the R form. In some
archaea the hydrophobic chains attached to the glycerol backbone are twice normal
length and pass completely through the membrane, attaching to a second backbone on
the opposite side. This adds extra stability to the membrane and these dual lipids are
often found in archaea living in extreme environments.

Figure 2-69 Ether-linked lipids

Archaea have an ether linkage between their fatty acids and the glycerol backbone,
which is in contrast to the ester linkage seen in bacteria and eukaryotes. (A) An ether-
linked lipid show as the chemical structure and a space-filling model. (B) A tetraether
lipid, showing a general chemical structure. An example of a tetraether lipid from the
thermophilic archaeal species Thermoplasma acidophilium is shown as a space-filling
model.
Cell walls

As we have discussed, almost all bacteria have cell walls. These walls contain
peptidoglycan, with N-acetyl muramic acid being the signature molecule for the
presence of peptidoglycan. Archaea are considerably more diverse in the composition of
their cell walls. They lack peptidoglycan, but some contain pseudomurein that has a
similar structure as shown in Figure 2-54 [140]. N-acetylalosaminuronic acid replaces
N-acetylmuramic acid in the backbone of the molecule and each glycan unit is linked
together using 1,3 glycosidic bonds, instead of the 1,4 glycosidic bonds seen in
peptidoglycan. Many archaea do not contain a peptidoglycan molecule in any form,
instead covering the outside of the membrane with proteins, glycoproteins or
polysaccharides. Scientists refer to these cell walls as S-layers. In any case the function
of the cell wall remains the same, containing the cytoplasm and giving the microbe its
shape.

Figure 2-54 A comparison of cell wall structure in archaea

Archaea have several different types of cell wall. Some contain a structure reminiscent
of peptidoglycan called pseudomurein. The chemical formula is pictured on the left.
Other microbes will have a surface layer (S-layer) composed of repeating units of one or
a few proteins, glycoproteins or sugar. These crystal lattices serve to protect the cell.
The micrograph on the right shows the surface of Methanospirillum hungatei cells. Note
the regular repetition of the pattern on the outside surface. (Source: M. Firtel, et al.
1993. J. Bacteriol. 175:7550-7560)

These various differences between archaea, bacteria and eukaryotes indicate major
differences between these life forms, and phylogenetic analysis bears out this
conclusion. It is clear that the archaea are more divergent from bacteria than we are
from an amoeba. This in turn indicates that the common progenitor of these groups
existed very early in evolution. Analysis and classification of the archaea has
fundamentally changed how biologists think about organismal diversity.

2 - 39 Eukaryotic cells have much in common with prokaryotic cells

  There are many basic similarities between eukaryotic and prokaryotic cell
structures, but some structures are different.
 Many enzymes that carry out the same function between eukaryotes and bacteria
are homologous.
 The basic structure and function of DNA is similar, but eukaryotes have a more
highly organized genome that organizes itself into chromosomes during cell
division.
 The overall sequence of transcription and translation is very similar, although
the details can be different.
 Membranes are highly conserved throughout all life forms.

After the recent journey through the bacterial cell, you may have started to wonder
about your own cells or other eukaryotic cells. How many properties do we share with
bacteria? How are we different? It turns out, as you might expect, we share some basic
things in common, but other structures are very different.

It turns out that much of what you now know about bacterial cells also applies to those
of eukaryotes. It is safe to say we are more similar than we are different. The basic
building blocks of the cell, such as nucleic acids, amino acids and sugars are identical.
Macromolecular organizations such as chromosomes and membranes have many
similarities. Many proteins in eukaryotes, especially those that carry out essential cell
functions, have homologs in bacteria that share a high degree of sequence and structural
similarity. An example that illustrates this point is the respiratory enzyme cytochrome
oxidase. As shown in Figures 2-55 and 2-61, a comparison of cytochrome oxidase from
the bovine and Rhodobacter sphaeroides reveals a near identical arrangement of the
catalytic proteins and high sequence similarity. However, the cytochrome oxidase in the
bovine has a number of other polypeptides that serve a structural role.

Figure 2-55 Comparison of cytochrome oxidase from bacteria and bovine.

Molecular models of cytochrome oxidase from Rhodobacter sphaeroides (A) and
bovine (B) are compared. Each protein is a complex of several distinct proteins, but the
four polypeptides shown in color have a high degree of similarity in both their sequence
(see Figure 2-61) and structure. The structural similarity should be obvious in this view.
Such structurally similarity cannot have arisen by chance, but must reflect the evolution
of each from a single ancestor. The gray polypeptides in the bovine cytochrome oxidase
are not found in the bacterial protein.

Figure 2-61 Sequence comparison of cytochrome oxidase from three

species
A sequence comparison of cytochrome oxidase showing the high degree of identical
amino acids between these very different species: cows and two different bacteria. The
colored boxed indicate where the amino acids are identical or similar among the three
sequences and the different colors refer to different classes of amino acids.

The DNA in all organisms is chemically similar but the organization of the helix into
higher order structures varies. Eukaryotes contain a larger number of histones
(chromosome-binding proteins) and in many cases during cell division, compaction of
the chromosome takes place, so that they are visible using light microscopy.

The basic mechanism of converting genetic information into proteins is also rather
similar. Most all of the central components of this process show sequence similarities
across biology, and substantial functional similarities as well. The fact that eukaryotes
have nuclei means that mRNA must be transported outside of that structure before
translation can begin, but the process is otherwise rather similar.

The most conserved of all structures is probably the membrane. Membranes enclose all
living systems and every membrane contains amphipathic lipids. The exact chemical
structure of the lipids is often different depending upon the species and its environment,
but the overall arrangement of the membrane is the same. Membranes perform
remarkably similar functions in all species: keeping the cytoplasm in and the
environment out. Cholesterol is common in the membranes of eukaryotes, but is
uncommon in bacterial and archaeal membranes.

2 - 40 Things that are different between eukaryotes and prokaryotes

 Eukaryotes contain organelles that perform various specific functions for the
cell.
 A highly organized cytoskeleton is present in eukaryotes.

Eukaryotes are typically more complex than prokaryotes and appear more organized
when examined in the microscopic. This organization into different intracellular
compartments likely reflects the demands of a more complex cellular system. It may
also be that the formation of these separate organelles allowed the subsequent evolution
of more elaborate cells. Figure 2-70 [141] diagrams the various structures found in the
typical eukaryotic cells. Note that chloroplasts are only found in photosynthetic
organisms. The organelles are the nucleus [142], the mitochondria [143], the
endoplasmic reticulum [144] and the golgi apparatus [145]. Internally eukaryotic cells
have a cytoskeleton that determines cell structure and in plants this is supplemented by
cell walls. We discuss these structures and their functions in eukaryotic cells in the
following sections.

Figure 2-70 Eukaryotic cell structure

A diagram of the common structures found in eukaryotic cells.

2 - 41 Unique structures in eukaryotes

 Microfilaments and microtubules form a network of fibers that determine cell

shape, participate in cell division, move organelles around the cell and form
motility structures.
 The endoplasmic reticulum is a network of tube and vesicles involved in
membrane synthesis and in export of protein and other substances from the cell.
 The Golgi apparatus is a double membrane structure mainly concerned with the
maturation of proteins synthesized in the endoplasmic reticulum and the
formation of lysosomes.
 Lysosomes are membrane encircled structures that contain digestive enzymes.
Their exact role in the cell depends upon the cell type.

Microfilaments and microtubules

The cytoskeleton is a network of filaments and fibers found in the cytoplasm of many
eukaryotic cells. It serves four known roles in cells.

 The cytoskeleton helps to determine the shape of the cell.

 It is involved in cell division, allowing the separation of the chromosomes into
the daughter cells.
 The cytoskeleton moves organelles around the cell.
 It is involved in motility either through the use of flagella or by amoeboid
movement.

The cytoskeleton components can be divided into three classes based upon the size,
distribution and function of the filaments. Microfilaments are the smallest at 4 to 6 µm
in diameter and are made of actin. These lie beneath the surface of the cell membrane
and are anchored to it, forming a web inside the cell. They dictate the cell's shape and
can also be involved in motility by contraction or expansion of the filament. Filaments
may also tether organelles to the membrane and help move them around the cell. This
movement can be important for modulation of organelle function. Intermediate
filaments are 10 µm in diameter and are made of keratin, which is the same protein
found in hair and fingernails. These filaments take different forms and are found in
many types of cells, but their exact function is unknown. They may play a structural
role similar to that of some microfilaments. Figure 2-56 [146] shows some examples of
cytoskeletal elements

Figure 2-56 Cytoskeletal elements

Eukaryotic cells have several different types of scaffolding proteins to help them keep
their shape. Microfilaments, microtubules, and centrioles are all important structural
elements.

In addition to the above, there are more complex fibers and structures. Microtubules are
hollow cylindrical structures that are 20-25 µm in diameter containing tubulin as the
major structural protein. Tubulin polymerizes into a helical cylindrical structure and
thirteen of these protofilaments then combine to make a microtubule. Microtubules can
form the basis of a number of different structures. Many of these structures form to
perform a necessary function and are then disassembled afterwards. Often microtubules
form centrioles that contain nine sets of microtubules arranged in a circular matrix.
These are 400 µm long and 150 µm wide and are usually found in pairs at right angles
to each other. Centrioles are important in proper chromosome segregation and cell
division as discussed in the section on the nucleus below. Microtubules are also part of
the basal bodies of flagella and cilia.

Cilia and flagella are examples of more permanent structures that contain microtubules.
Both cilia and flagella have a similar structure of nine pairs of microtubules arranged in
a circular fashion around a tenth pair that runs down the center. Both are attached to the
membrane and project into the environment. In a process that requires energy, the
microtubules in the outer ring are moved with respect to each other, causing the cilia or
flagella to bend and snap back in a whip-like fashion. This bending causes the
movement of liquid near the structures such that spent liquid with few nutrients and
waste products is moved away from the cell and is replaced by fresh liquid containing
nutrients and oxygen. The beating of cilia and flagella can also push the cell through its
environment. Cilia are 2 to 10 µm long and 0.5 µm wide and are shorter and typically
more numerous than flagella with hundreds of them on some types of cells. An example
of a ciliated organism is the unicellular protist Paramecium, which can be found in fresh
water ponds. The microbe is a predator of bacteria and motility is vital in this life-style,
both for chasing down prey and moving away from danger. Cilia cover the surface of
Paramecium and move the organism through the environment by beating in a
coordinated fashion. Figure 2-57 [147] shows one example of a protozoan.

Flagella are 50-100 µm in length and there are typically only one or two per cell.
Eukaryotic flagella are larger than those found on bacteria or archaea and have a more
complex structure. Flagella are found in many unicellular creatures with one example
being the dinoflagellates and their primary role is cell motility. These aquatic creatures
contain two flagella; one encircling the body of the organism while the other is attached
in a perpendicular fashion to the first. Dinoflagellates are often photosynthetic and
important as primary producers in the oceans.

Endoplasmic reticulum

The endoplasmic reticulum (ER) is a finely divided system of interconnected

membranes, consisting of tubules and vesicles that loop through the cell and are
contiguous with the nuclear membrane. A drawing of the ER is shown in Figure 2-58
[148]. It functions in the synthesis of membranes and membrane proteins and is also
involved in protein secretion. Not surprisingly, the ER is especially prominent in cells
doing a large amount of protein secretion. The ER works very closely with the Golgi
apparatus (see below) to carry out these functions. There is no structure in bacterial
cells that is analogous to the ER, but many of the same functions are carried out on the
inside surface of the cellular membrane in bacteria. ER comes in two types: rough ER
and smooth ER.

Figure 2-58 The endosplasmic reticulum

Eukaryotic cells contain a network of passages that connect various elements of the cell
and are also important in secretion.

Rough ER gets its appearance from the presence of ribosomes on its surface as seen in
the electron microscope, and its function is the production, processing and export of
proteins. During translation an appropriate signal guides the ribosome to the ER
membrane and causes the protein to be synthesized directly across the membrane into
the lumen of the ER. There proteins may be processed or modified by the addition of
carbohydrates to form glycoproteins. After processing proteins move slowly through the
ER and are packaged into vesicles of ER membrane called transition vesicles. These
release from the ends of the ER and move by elements of the cytoskeleton either to the
Golgi apparatus or to the plasma membrane. Once contact is made between the
transition vesicle and the Golgi or the plasma membrane, the two fuse and release the
contents of the vesicle into the target compartment.

Smooth ER does not contain ribosomes and the lumen and membrane of smooth ER
contain a variety of enzymes that perform many functions including modification of
toxins and synthesis of steroids.

Golgi apparatus

The Golgi apparatus is an organelle containing a double membrane and it is mainly

devoted to the processing of proteins synthesized in the ER. A drawing of the Golgi
apparatus is shown in Figure 2-59 [149]. It is found in many eukaryotic cells, but it lacks
a well-formed structure in many fungi and ciliate protozoa. It consists of regions of
stacked contiguous membranes containing no ribosomes. Each membrane sac is 15 to
20 µm thick and separated from the next stack by about 30 µm. A complex network of
tubes and vesicles extend from the edges of these sacs into the surrounding cytoplasm.
The stack of membranes has a definite polarity with those near the ER (the cis face)
having a different shape and enzyme content than those at the opposite end (the trans or
maturing face). Studies of the Golgi apparatus appear to show material flowing into the
cis face from vesicles, through the apparatus and then exiting at the trans face.

Figure 2-59 The Golgi Apparatus

The Golgi apparatus is involved in the glycosylation and proteolytic processing of
proteins that are to be secreted into various cellular organelles or to the outside
environment. Proteins often pass through the Golgi apparatus as part of their
maturation.

The role of the Golgi apparatus is to package material for export, but its exact function
varies depending upon the organism. For example, Giardia and Entamoeba utilize
Golgi apparatus to form cell walls during cyst formation. It often participates in the
synthesis of cell membranes and the final processing of proteins before export. In many
cases the Golgi apparatus contains glycosylation enzymes [150] that add sugars to
proteins as they move through its lumen. The type of glycosylation that takes place is
dependent upon signals contained within the protein sequence. The Golgi also processes
enzymes using proteases, which clip at specific amino acids sequences to form mature
proteins and hormones. These mature proteins then move to their final destinations,
which may be in the membrane, in lysosomes or secreted into the environment.

Lysosomes

One of the most important functions of the Golgi apparatus is the synthesis of
lysosomes [151], an organelle that is found in a variety of eukaryotic cells. Lysosomes
are spherical structures enclosed in a single membrane that can vary in size from 50 µm
to several µm. They are involved in intracellular digestion and contain enzymes (called
hydrolases) that digest many types of macromolecules. Hydrolases function best under
acidic conditions (pH 3.5 to 5.0) and the lysosome maintains this pH by membrane
proteins that pump protons into its interior. Enzymes bound for lysosomes are
synthesized on ribosomes that deposit them in the rough ER. They then move though
the smooth ER and Golgi apparatus before being package into lysosomes. In some cases
lysosomes can also bud off from the smooth ER.

Lysosomes serve a variety of functions depending upon the cell type. In unicellular
eukaryotes they are often digestive structures that take bacteria or other substances from
the outside environment and degrade them into usable nutrients. In mammals,
lysosomes serve to eliminate unwanted particles, either cell structures that are no longer
needed or foreign macromolecules (from viruses or bacteria) that have invaded the cell.
In any case the hydrolytic enzymes and low pH typically inactivate and then degrade
any particle that enters the lysosome

2 - 42 Eukaryotic cells absorb things by endocytosis

 The major pathway into eukaryotic cells is by endocytosis. This process takes
two forms, phagocytosis and pinocytosis.
 The Golgi apparatus, ER, lysosomes and endocytosis work as a coordinated
whole to move particles into and out of the cell.

Lysosomes are an important part of endocytosis, a process that eukaryotic cells use to
take up particles from the environment. Endocytosis is illustrated in Figure 2-60 [152].
The process creates membrane-bound cavities filled with fluid and solid materials.
Larger membrane-enclosed cavities are called vacuoles while smaller ones are called
vesicles. Endocytosis comes in two forms, phagocytosis and pinocytosis. Phagocytosis
involves the engulfment of large particles, even microorganisms, into membrane-bound
compartments. It is a process used most often in the immune system and is described in
detail in the chapter on infection and immunity. Pinocytosis involves the recognition of
specific particles in the environment as described below. The process is used by
unicellular eukaryotic microbes to ingest food and by multicellular organisms to take in
certain macromolecules traveling from other parts of the organism.

Figure 2-60 The process of endocytosis

Eukaryotic cells absorb materials from the outside by using endocytosis. This is a
standard pathway through which most material enters the cell.

Pinocytosis begins when protein receptors on the cell surface bind to the target molecule
to be ingested. The bound particle migrates to an area of the membrane that is rich in a
protein called clathrin. This protein forms a matrix and causes an indentation in the
membrane called a clathrin-coated pit. The entrance of a receptor with a bound particle
begins a process of invagination at the pit that results in the internalization of the pit and
the engulfment of the incoming particle inside a membrane vesicle. This vesicle is
called an endosome and once inside the cell, proteins in the endosome membrane begin
to pump protons inside, dropping the internal pH of the endosome. The clathrin on the
membrane of the endosome then migrates back to the plasma membrane to repeat the
cycle. The endosome fuses with a lysosome to form an endolysosome, which causes the
ingested particle to be degraded. The valuable breakdown products are transported out
of the endolysosome into the cell. The spent endolysosome is called a residual body and
sometimes fuses with the plasma membrane to release remaining compounds into the
environment.

The ER, Golgi apparatus, lysosomes and endosomes seem to operate as a coordinated
whole, functioning in the import and export of materials. It is probably correct to think
of these structures as a functional unit and the term vacuome [153] has been coined to
describe them. Ribosomes in the ER manufacture proteins and these are modified in the
ER and Golgi. The mature proteins eventually find their way to the plasma membrane
for export or become part of lysosomes. Lysosomes serve in the endocytotic pathway to
take up materials and process them for cell use. They also digest spent cell constituents.
All of these processes occur within membrane structures and this carefully controls the
import and export of materials from the cell. Considering the extent of these structures
in the cell it is remarkable that the membranes of these structures, especially lysosomes,
never rupture. If they did it would be catastrophic to the cell and would rapidly lead to
cell death.

2 - 43 The nucleus holds the cells genetic material in eukaryotes

 The genome of eukaryotes is sequestered to a membrane bound organelle called

the nucleus.
 The nucleus is the site of replication and transcription.
 Most eukaryotes have more than one chromosome in their nucleus and
replication of these chromosomes proceeds through a sequence of steps that are
visible with a light microscope.
 The nucleus contains visible spots called nucleoli, which are the location or
ribosome synthesis.

While the bacterial cell does seem to sequester its chromosome to a portion of the
cytoplasm, there is no demarcation that divides the nucleoid from the rest of the cell. In
eukaryotes the nuclear membrane separates the cell's DNA from the cytoplasm. The
nucleus is the largest and most clearly visible organelle of eukaryotic cells. It contains
almost all the cell's DNA and is the site of chromosome replication and transcription. It
has two layers of membrane encircling it called the nuclear envelope, with the outer
layer being contiguous with the ER. Scattered throughout this nuclear envelope are
circular openings known as nuclear pores. These pores are highly discriminatory,
allowing easy movement in and out of the nucleus of only appropriate macromolecules
such as proteins with specific sequences.

In eukaryotes, the chromosome is not a single circular piece of DNA as in most

prokaryotes. Rather, it is split into a number of linear chromosomes with each cell
containing at least two copies of each chromosome. The exceptions are those cells that
specialize in reproduction and only contain one copy of the cell's chromosomes. Each
piece of DNA is complexed with special basic structural proteins called histones that
seem to be important in keeping the DNA organized. The DNA is also bound by other
proteins involved in its maintenance and the entire set of DNA and associated proteins
is called chromatin. For much of the cell cycle chromatin consists of long DNA strands
formed into beads by association of histones along it length. Figure 2-71 [154] shows a
nucleus in the midst of mitosis, with the chromosomes visible.

Figure 2-71 A eukaryotic cell in the middle of mitosis

Eukaryotic DNA replication takes place during the cell phase called mitosis. At this
time, protein synthesis is halted and the chromosomes condense. The sister chromatids
meet at the middle of the cell and then migrate to two separate poles. This movement is
coordinated by centrosomes, kinetochores and microtubules.
In actively growing cells the DNA is replicated from numerous sites, rather than the
single bi-directional origin in prokaryotes. This is necessary due to the much larger
amount of DNA found in most eukaryotic cells. During division in prokaryotes, the cell
appears to simply split in two with each daughter cell receiving a chromosome. In
contrast, eukaryotic cells go through a morphologically distinct phase, mitosis, to
achieve separation of the chromosomes. One of the more important events of mitosis is
the binding of additional histones and the contraction of the chromatin into compact
structures that were called chromosomes due to their staining properties. (The original
meaning of the term chromosomes is a colored body, but is now synonymous with a
cell's DNA). The two daughter chromosomes formed during replication are physically
separated into the separate daughter cells by the filaments called microtubules [155].
These attach at one end to the chromosome at a region termed the kinetochores and at
the other end they attach to one of two regions of the cell called a centrosome [156]. By
depolymerization of the microtubules at each centrosome, each daughter chromosome is
pulled away from its partner and toward a region that eventually reforms as a new
nucleus.

There are also a number of important differences in transcription between eukaryotes

and prokaryotes. In eukaryotes, mRNA transcription takes place in the nucleus and the
finished mRNA moves through the nuclear pores and into the cytoplasm for translation
by the ribosomes. The genes of eukaryotic cells also contain regions of largely
unimportant DNA, termed introns [157], that do not code for protein. After a gene is
transcribed into mRNA these introns are removed before translation. One set of nuclear
proteins removes these sequences and splices the actual coding sequences (exons)
together to make the finished mRNA. The finished mRNA then travels from the nucleus
to ribosomes in the cytoplasm. The mRNA of eukaryotic cells is also decorated with
modifications at each end that affect the stability of the mRNA. At the front end is
usually a 5�-cap made of 7-methylguanine attached to the mRNA by a triphosphate
linkage. At the 3' end of the mRNA is a long stretch of A bases (a poly-A tail) that have
a role in mRNA stability as mentioned earlier in this chapter. Finally, while it is quite
common in bacteria to have a number of genes on each mRNA transcript, the vast
majority of mRNAs in eukaryotes code for only a single protein product. Figure 2-72
[158] shows the steps in gene expression in eukaryotes.

Figure 2-72 The steps of gene expression in eukaryotes

Processing genetic information into protein is more complex in eukaryotes than in
prokaryotes. In part this is due to the existence of introns in most genes of "higher
eukaryotes" (though introns are rare in yeast). These have to be removed, a poly-A tail
added to the 3' end and a cap added to the 5' end of the mRNA before it reaches the
cytoplasm for translation.

Eukaryotic cells also contain one or more dark-staining structures within the nucleus
called nucleoli. Although they are not enclosed within a separate membrane the nucleoli
are complexes with separate granular and fibrillar regions. They are present in non-
dividing cells, but frequently disappear during mitosis and again reappear after cell
division is over. The nucleolus is the initial site of ribosome synthesis. This structure
contains the DNA that codes for the ribosomal RNA genes. The ribosomal RNAs are
synthesized and then processed to form the final rRNA molecules. These are then
combined with several ribosomal proteins synthesized in the cytoplasm to form an
initial ribosomal complex. The entire complex then migrates out of the nucleus into the
cytoplasm where it combines with other proteins to form a ribosome.

2 - 44 Mitochondria and plastids are organelles of energy generation in

eukaryotic cells

 Mitochondria are found in almost all eukaryotic cells and convert high-energy
electrons into ATP.
 Plastids are factories for photosynthesis, converting light energy into high-
energy electrons and ATP.
 Both of these structures trace their ancestry back to free-living prokaryotes.

Mitochondria are involved in energy generation through respiration. Mitochondria have

no fixed shape, but often look like short rods in transmission electron micrographs when
viewed along their long axis (Figure 2-73). Each mitochondrion contains two
membranes. The outer membrane is smooth and serves as a selective barrier. The inner
membrane is highly convoluted and folded and contains high numbers of membrane
complexes. Nutrients are oxidized inside the mitochondria by catabolic enzymes and the
high-energy electrons extracted are donated to a respiratory chain in the inner
membrane. These enzymes then create a proton gradient and this gradient is then used
to synthesize ATP. ATP leaves the mitochondria and it serves as a source of energy for
the rest of the cell's machinery.

Figure 2-73 Mitochondria structure

Mitochondria are rod-shaped structures that resemble the shape of common bacteria.
They contain two membranes, similar to what is found in gram-negative bacteria, and
70S ribosomes. Energy generation occurs at the inner membrane.

Plastids are specialized organelles involved in metabolism that are unique to plants and
come in several forms. Amyloplasts are starch storage containers found in some plants.
Chloroplasts are oval-shaped structures inside of plant and algal cells that contain an
outer and inner membrane as shown in Figure 2-74 [159]. The outer membrane serves a
similar function to the outer membrane of mitochondria, while the inner membrane
consists of a network of stacks of membranous disks, called thykaloids, which are
attached together by narrow tubes of membrane. The thykaloid membranes are the
centers of photosynthesis in eukaryotes. They contain enzyme complexes that capture
light and produce ATP and high-energy electrons that are used to form sugar from
carbon dioxide.

Figure 2-74 Chloroplast structure

Chloroplasts are the site of the light reactions of photosynthesis. Light striking the
chloroplast is converted into a proton motive force and this is used to generate energy.
Chloroplasts contain two membranes, again similar to gram-negative bacteria, and the
chloroplast itself is a relative of cyanobacteria.

Mitochondria and chloroplasts each have a single circular chromosome and large
numbers of ribosomes that are of bacterial (70S) and not eukaryotic (80S) form. The
presence of two lipid bilayers, a circular chromosome and 70S ribosomes is consistent
with the evolutionary hypothesis that we have already explained [160] near the
beginning of this chapter.

2 - 45 The implications of eukaryotic structures on their growth

 Eukaryotic cells are generally bigger than prokaryotic cells. This allows
eukaryotes to have organelles, have slower turnover rates of macromolecules,
and requires the presence of mechanisms to move things around the cell.
 The division of the chromosome from the rest of the cell and the larger size of
eukaryotes, slows the rate at which they can replicate.
 The presence of organelles, which are sensitive to harsh conditions, might limit
the extreme environments in which eukaryotes can grow.

Eukaryotic cells are generally much larger than prokaryotic ones and this difference in
volume has several implications. First bigger cells can afford to have more things stored
in the cytoplasm. This means it is not as costly to a eukaryotic cell to have structures
taking up space. In a prokaryote, space is at a premium and anything not being used is
pretty rapidly degraded. This may be one reason that organelles are possible. Second,
larger cells have a lower surface-to-volume ratio than do smaller cells and therefore
prokaryotes effectively have more contact with their environment. This greater exposure
can mean a more rapid response to changing environmental conditions. Finally bigger
cells have more of a challenge moving molecules within themselves. Prokaryotes can
often depend on simple diffusion to move molecules around the cell, but this process
might be too slow and inefficient in much larger cells. Eukaryotes overcome this by
having specific transport mechanisms (i.e. microtubules) inside the cell.

Size constrains eukaryotes in two important ways: how fast they can grow and what
environments they can tolerate. The compartmentalization of the genome inside the
nucleus limits the rate at which eukaryotic cells can divide. The complete cell division
cycle in a multicellular eukaryotic organism depends upon the cell type, but even in
rapidly dividing skin cells it takes at least 8 hours. In unicellular yeast cultures, the
shortest cell cycle is about 1.7 hours under ideal conditions. Due to their smaller
genomes, lack of a nucleus and the ability to couple transcription and translation,
bacteria can grow much faster. Clostridium perfringens has been shown to go through a
complete division cycle in at little as 6.6 minutes at 43 °C in beef cubes!

There are environments that have one or more physical characteristics that prevent the
growth of most organisms. These extreme environments might be too hot, cold, acidic
or alkaline for typical organisms to grow. However, a small subset of prokaryotes has
evolved to take advantage of these environments and thrive, and prokaryotes always
define the extremes of where life can exist. In part this is probably due to the fact that
simpler cells have fewer "body parts" that must be changed in order for growth under
very different conditions. In eukaryotes, they would need to modify not only their
cytoplasmic contents to tolerate the extreme environment, but also the makeup of their
organelles.

2 - 46 Summary
We have moved from within prokaryotic and eukaryotic cells to the outside, and this
should provide a framework for thinking about the make-up of microbes. Some ideas
that you should take away from this chapter are:

 Although prokaryotes are small, they have a huge amount of molecular

complexity. In a sense, multicellular organisms deal with different problems by
forming different cell types, but prokaryotes must adapt each cell to specific
environmental conditions. They are stunningly clever little machines.
 Cell structures are made up of polymers.
 These polymers are made from just a few basic types of molecules: amino acids,
sugars, nucleic acids and fatty acids.
 The sequence of the polymers dictates their structure and subsequently their
function.
 While not obvious in the microscope, the prokaryotic cell is organized into
functional units: (i) The nucleoid organizes, manages and replicates the DNA.
(ii) The cytoplasm carries out many of the reactions of the cell including
biosynthesis and catabolism. (iii) The ribosomes are the site of protein synthesis.
(iv) The membrane defines the borders of the cell and serves as a barrier to most
molecules. (v) The cell wall defines a microbes shape and helps protect it from
the environment.
 The building blocks that make up the average prokaryote are also found in all
living things on this planet. We share more things in common with microbes
than there are features that distinguish us.

In subsequent chapters we will learn what all these various parts are doing.