Exploring the Microbial World - Microorganisms, Tiny Titans of the

Earth

 Microorganisms—those organisms too small to be seen clearly by the unaided

eye, are fabulously diverse and unimaginably abundant. It is difficult to count the
number of microbes on Earth, but estimates are about 10 30 microbial cells in
habitats as diverse as termite guts and sediments deep beneath the seafloor.
 There are more microbes on Earth than stars in the known universe. Although
microbes are generally 1 millimeter or less in diameter, some, such as bread
molds, are visible without microscopes.
 Some macroscopic microorganisms are multicellular. They are distinguished
from other multicellular life forms such as plants and animals by their lack of
highly differentiated tissues.
 In addition, a variety of acellular biological entities, including viruses and sub-
viral agents, are also called microorganisms and microbes. This is not without
controversy because these agents cannot reproduce independently.
 Cells that came to be called prokaryotic cells (Greek pro, before; karyon, nut or
kernel) have an open floor plan. That is, their contents are not divided into
compartments by membranes.
 Only eukaryotic cells (Greek eu, true) have a nucleus and other membrane-
bound organelles (e.g., mitochondria, chloroplasts) that separate some cellular
materials and processes from others.
 These observations eventually led to the development of a classification scheme
that divided organisms into five kingdoms: Monera, Protista, Fungi, Animalia,
and Plantae. Microorganisms (except for viruses and other acellular infectious
agents) were placed in the first three kingdoms. In this scheme, all organisms
with prokaryotic cell structure were placed in Monera.
 However, the five-kingdom system is no longer accepted by microbiologist.
This is because prokaryotes are too diverse to be grouped together in a single
kingdom.
 Classifying microbes has benefited from progress in three areas.
 First, the development of electron microscopy techniques reveals the detailed
structure of microbial cells.
 Second, methods that measure the biochemical and physiological characteristics
of many different microorganisms demonstrate many similarities and differences.
 Third, the genomic revolution enabled the analysis of nucleic acid and protein
sequences from a wide variety of organisms.
 The comparison of ribosomal RNA (rRNA) nucleic acid sequences, begun by
Carl Woese (1928–2012) in the 1970s, transformed our understanding of the
term prokaryote.
 It was discovered that there are two very different groups of organisms with
prokaryotic cell morphology: Bacteria and Archaea.
 Among eukaryotic microbes, later studies showed that Protista is not a cohesive
taxonomic unit (i.e., taxon) and that it should be divided into three or more
kingdoms.
 These studies and others led many taxonomists to reject the five-kingdom system
in favor of one that divides cellular organisms into three domains: Bacteria,
Archaea, and Eukarya (all eukaryotic organisms)

 Bacteria

 Members of domain Bacteria are usually single-celled organisms.

 Most have cell walls that contain the structural molecule peptidoglycan.
 Most bacteria do not cause disease. In fact, bacteria are major inhabitants of our
bodies, forming the human microbiome. Indeed, at least as many microbial cells
found inbody digest food and produce vitamins. In these and many other ways,
the human microbiome helps maintain our health and well-being.
 Unfortunately some bacteria do cause disease, and some of these diseases can
have a huge impact. In 1347 the plague (Black Death), a disease caused by
bacteria living in fleas, struck Europe with brutal force, killing one-third of the
population within 4 years. Over the next 80 years, the disease struck repeatedly,
eventually wiping out roughly half of the European population. The resulting
labor shortage gave workers more power, ultimately eliminating serfdom, and
preparing the way for the Renaissance.

 Archaea
 Members of domain Archaea are distinguished from bacteria by many features,
most notably their distinctive rRNA sequences, cell walls, and membrane
lipids. Some have unique metabolic characteristics, such as the ability to generate
methane (natural) gas.
 Some archaea are found in extreme environments, including those with high
temperatures (thermophiles) and high concentrations of salt (extreme
halophiles).
 Archaea do not appear to directly cause disease in humans.

 Eukarya

 Domain Eukarya includes plants, animals, and microorganisms classified as

protists or fungi.
 Protists are generally unicellular but larger than most bacteria and archaea. They
have traditionally been divided into protozoa, which have an animal-like
metabolism, and algae, which are photosynthetic.
 However, these terms lack taxonomic value because protists, algae, and protozoa
do not form three groups, each with a single evolutionary history. Nonetheless,
for convenience, we use these terms here. Protist diversity reflects broad
phylogeny.
 Fungi are a diverse group of microorganisms that range from unicellular forms
(yeasts) to multicellular molds and mushrooms. Because of their metabolic
capabilities, many fungi play beneficial roles, including making bread dough rise,
producing antibiotics, and decomposing dead organisms.
 Some fungi associate with plant roots to form mycorrhizae. Mycorrhizal fungi
transfer nutrients to the roots, improving growth of the plants, especially in poor
soils. Other fungi cause plant diseases (e.g., rusts, powdery mildews, and smuts)
and diseases in humans and other animals.

 Virus

 The microbial world also includes numerous acellular infectious agents.

Viruses are acellular entities that must invade a host cell to multiply. The
simplest virus particles (also called virions) are composed only of proteins and a
 nucleic acid, and can be extremely small (the smallest is 10,000 times smaller
than a typical bacterium).
 However, their small size belies their power. They cause many animal and plant
diseases and, as we saw most recently with COVID-19, can trigger epidemics and
pandemics that shape human history. In addition to COVID-19, viral diseases
include rabies, influenza, AIDS, the common cold, and some cancers.
 Viruses are also important in aquatic environments, where they play a critical role
in shaping microbial communities.
 Viroids are infectious agents composed only of ribonucleic acid (RNA). They
cause numerous plant diseases.
 Satellites are composed of a nucleic acid enclosed in a protein shell. They must
co-infect a host cell with a virus, called a helper virus, to complete their life
cycle. Satellites and their helper viruses cause both plant and animal diseases.
 Finally, prions, infectious agents composed only of protein, are responsible for
causing neurological diseases such as scrapie and “mad cow disease.”
MICROBES HAVE EVOLVED AND DIVERSIFIED FOR BILLIONS OF
YEARS
 Theories of the Origin of Life Depend Primarily on Indirect Evidence
 Dating meteorites through the use of radioisotopes places our planet at an
estimated 4.5 to 4.6 billion years old.
 However, conditions on Earth for the first 100 million years or so were far too
harsh to sustain any type of life. Eventually bombardment by meteorites
decreased, water appeared on the planet in liquid form, and gases were released
by geological activity to form Earth’s atmosphere.
 These conditions were amenable to the origin of the first life forms. But how did
this occur, and what did these life forms look like?
 Most definitions of life consist of a set of attributes. The attributes of particular
importance to paleobiologists are an orderly structure, the ability to obtain and
use energy (i.e., metabolism), and the ability to reproduce.
 Just as NASA scientists are using the characteristics of microbes on Earth today
to search for life elsewhere, so too are scientists examining extant organisms,
those organisms present today, to explore the origin of life.
 Some extant organisms have structures and molecules that represent relics of
ancient life forms.
 The best direct evidence for the nature of primitive life would be a fossil record.
There have been reports of microbial fossil discoveries since 1977. These have
always met with skepticism because some objects that look like cells can be
formed by geological forces that occurred as the rock was formed. The result is
that the fossil record for microbes is sparse and always open to reinterpretation.
 Despite these problems, most scientists agree that life was present on Earth about
3.5 to 3.8 billion years ago. To reach this conclusion, biologists rely on indirect
evidence.
 Among the indirect evidence used are molecular fossils. These are chemicals
found in rock or sediment that are chemically related to biological molecules. For
instance, the presence of molecules called hopanes in a rock indicates that
bacteria were present when the rock was formed. This conclusion is reached
because hopanes are formed from hopanoids, which are found in the plasma
membranes of extant bacteria.

 Early Life Was Probably RNA-based

 Before there was life, most evidence suggests that Earth was a very different
place: hot and anoxic, with an atmosphere rich in water vapor, carbon dioxide,
and nitrogen. In the oceans, hydrogen, methane, and carboxylic acids were
formed by geological and chemical processes.
 Areas near hydrothermal vents may have provided the conditions that allowed
chemicals to react with one another, randomly testing the usefulness of the
reaction and the stability of its products.
 Some reactions generated molecules that functioned as catalysts, some
aggregated with other molecules to form the predecessors of modern cell
structures, and others were able to replicate and act as units of hereditary
information.
 In modern cells, three different molecules fulfill the roles of catalysts, structural
molecules, and hereditary molecules. Proteins have two major roles in modern
cells: catalytic and structural.
 Catalytic proteins are enzymes and structural proteins serve myriad functions,
such as transport, attachment, and motility.
 DNA stores hereditary information that is replicated and passed on to the next
generation.
 RNA converts the information stored in DNA into protein.
 Proteins can do cellular work, but their synthesis involves other proteins and
RNA, and uses information stored in DNA.
 DNA cannot do cellular work, and proteins are needed for its replication.
 RNA synthesis requires both DNA as the template and proteins as catalysts.
 Based on these considerations, it is hypothesized that at some time in the
evolution of probionts, there must have been a single molecule that could do
both cellular work and replicate.
 This idea was supported in 1981 when Thomas Cech discovered an RNA
molecule in a protist (Tetrahymena sp.) that also had catalytic activity. Since
then, other catalytic RNA molecules have been discovered, including an RNA
found in ribosomes that is responsible for forming peptide bonds—the bonds that
hold together amino acids, the building blocks of proteins.
 Catalytic RNA molecules are now called ribozymes. The discovery of ribozymes
suggested that RNA at some time was capable of storing, copying, and
expressing genetic information, as well as catalyzing other chemical
reactions.
 In 1986 Nobel laureate Walter Gilbert coined the term RNA world to describe
this pre-cellular stage in the evolution of life.
 However, for this pre-cellular RNA-based stage to proceed to the evolution of
cellular life forms, a lipid membrane must have formed around the RNA.
 This important evolutionary step is easier to imagine than other events in the
origin of cellular life forms because lipids, major structural components of the
membranes of modern organisms, spontaneously form liposomes—vesicles
bounded by a lipid bilayer.
 Apart from its ability to perform catalytic activities, the function of RNA suggests
its ancient origin.
 The cellular pool of RNA in modern cells exists in the ribosome, a structure that
consists largely of ribosomal RNA (rRNA) and uses messenger RNA (mRNA)
and transfer RNA (tRNA) to construct proteins.
 Also rRNA itself catalyzes peptide bond formation during protein synthesis. Thus
RNA seems to be well poised for its importance in the development of proteins.
 Because RNA and DNA are structurally similar, RNA could have given rise to
double-stranded DNA. It is suggested that once DNA evolved, it became the
storage facility for genetic information because it provides a more chemically
stable structure.
 Other pieces of evidence support the RNA world hypothesis: the fact that the
energy currency of cells, ATP, is a ribonucleotide and the discovery that RNA
can regulate gene expression.
 The evolution of metabolism. The early Earth was a hot environment that lacked
oxygen. Thus cells that arose there must have been able to use the available
energy sources under these harsh conditions.
 Today there are heat-loving archaea capable of using inorganic molecules such
as FeS as a source of energy. Some suggest that this interesting metabolic
capability is a remnant of the first form of energy metabolism.
 Another metabolic strategy, oxygen-releasing photosynthesis (oxygenic
photosynthesis), appears to have evolved perhaps as early as 2.7 billion years ago.
This is supported by the discovery of ancient stromatolites.
 Stromatolites are layered rocks formed by the incorporation of mineral
sediments into layers of cyanobacteria growing in thick mats on surfaces. The
oxygen released by these early cyanobacteria is thought to have altered Earth’s
atmosphere to its current oxygen-rich state, allowing the evolution of additional
energy-capturing strategies such as aerobic respiration, the oxygen-consuming
metabolic process used by many microbes and animals.

 Evolution of the Three Domains of Life

 “Origin” indicate the last universal common ancestor (LUCA) to all three
domains should be placed.
 LUCA is the most recent organism from which all three types of life— bacterial,
archaeal, and eukaryotic—arose.
 On this tree of life, LUCA is on the bacterial branch, which means that Archaea
and Eukarya evolved independently, separate from Bacteria.
 The evolutionary relationship of Archaea and Eukarya is still a matter of
considerable debate. According to the universal phylogenetic tree, Archaea and
Eukarya shared common ancestry but diverged and became separate domains.
 Recent evidence supports the notion that Eukarya evolved from Archaea . The
close evolutionary relationship of these two forms of life is still evident in the
manner in which they process genetic information.
 For instance, certain protein subunits of archaeal and eukaryotic RNA
polymerases, the enzymes that catalyze RNA synthesis, resemble each other to
the exclusion of those of bacteria.
 However, archaea have other features that are most similar to their counterparts in
bacteria (e.g., mechanisms for conserving energy).

 Mitochondria, Mitochondria-Like Organelles, and Chloroplasts Evolved

from Endosymbionts

 The endosymbiotic hypothesis is generally accepted as the origin of several

eukaryotic organelles, including mitochondria, chloroplasts, and
hydrogenosomes.
 Endosymbiosis is an interaction between two organisms in which one organism
lives inside the other. The original endosymbiotic hypothesis proposed that over
time a bacterial endosymbiont of an ancestral cell in the eukaryotic lineage lost its
ability to live independently.
 If the intracellular bacterium used aerobic respiration, it became a
mitochondrion. If the endosymbiont was a cyanobacterium and therefore
performed photosynthesis, it became a chloroplast
 Although the mechanism by which the endosymbiotic relationship was
established is unknown, there is considerable evidence to support this hypothesis.
 Mitochondria and chloroplasts contain DNA and ribosomes; both are similar to
bacterial DNA and ribosomes.
 Peptidoglycan, the unique bacterial cell wall molecule, has even been found
between the two membranes that enclose the chloroplasts of some algae.
 More specifically, mitochondria are most closely related to bacteria called
proteobacteria.
 The chloroplasts of plants and green algae are thought to have descended from an
ancestor of the cyanobacterial genus Prochloron, which contains species that live
within marine invertebrates.
 The endosymbiotic hypothesis for mitochondria has been refined by the hydrogen
hypothesis.
 This asserts that the endosymbiont was an anaerobic bacterium that produced H2
and
 CO2 as end products of its metabolism. Over time, the host became dependent on
the H2 produced by the endosymbiont. Ultimately the endosymbiont evolved into
one of several organelles.
 Some endosymbionts evolved into organelles such as a hydrogenosome—an
organelle found in some extant protists that produces ATP by a process called
fermentation.

 Evolution of Cellular Microbes

 The ancestral bacteria, archaea, and eukaryotes possessed genetic information

that could be duplicated, lost, or mutated in other ways.
 Mutations could have many outcomes. Some led to the death of the microbe, but
others allowed new functions and characteristics to evolve.
 Mutations that allowed the organism to increase its rate of reproduction or
survival were selected and passed on to subsequent generations.
 In addition to selective forces, geographic isolation of populations allowed some
groups to evolve separately from others. Thus selection and isolation led to the
eventual development of new collections of genes (i.e., genotypes) and new
species.
 In addition to mutation, other mechanisms exist for reconfiguring genomes and
therefore creating genetic diversity.
 Most eukaryotic species increase their genetic diversity by reproducing sexually,
whereby each offspring has a mixture of parental genes and a unique genotype.
Bacteria and archaea do not reproduce sexually. They increase their genetic
diversity by mutation and horizontal gene transfer (HGT).
 During HGT, genetic information from a donor organism is transferred to a
recipient, creating a new genotype in the recipient. In this way genetic
information is passed between individuals of the same generation and even
between species found in different domains of life.
 Genome sequencing has revealed that HGT has played an important role in the
evolution of all microbial species.
 Importantly, HGT still occurs in bacteria and archaea leading to the rapid
evolution of microorganisms with antibiotic resistance, new virulence properties,
and novel metabolic capabilities.
 The outcome of HGT is that most microbes have mosaic genomes composed of
bits and pieces of the genomes of other organisms.
MICROBIAL CULTIVATION EXPANDS THE HORIZON OF
MICROBIOLOGY

 Following the discovery of microorganisms driven by microscopic methods,

major discoveries in microbiology were fueled by advances in microbial
cultivation.
 Even before microorganisms were seen, some investigators suspected their
existence and role in disease. Among others, the Roman philosopher Lucretius
(about 98–55 bce) and the physician Girolamo Fracastoro (1478–1553)
suggested that disease was caused by invisible living creatures.
 However, until microbes could actually be seen and studied in some other way,
their existence remained a matter of conjecture.
 Therefore microbiology is defined not only by the organisms it studies but also by
the tools used to study them.
 The development of microscopes was the critical first step in the evolution of
the discipline.
 A distinct feature of microbiology is that microorganisms are usually removed
from their normal habitats and grown in isolation, apart from all other microbes.
This is called a pure or axenic culture.
 Although the development of techniques for isolating microbes in pure
culture was another critical step in microbiology’s history, it is now recognized
as having limitations.

 Microscopy Led to the Discovery of Microorganisms

 The earliest microscopic observations of organisms appear to have been made

between 1625 and 1630 on bees and weevils by the Italian Francesco Stelluti
(1577–1652), using a microscope probably supplied by Galileo (1564–1642).
 Robert Hooke (1635–1703) is credited with publishing the first drawings of
microorganisms in the scientific literature. In 1665 he published a highly detailed
drawing of the fungus Mucor in his book Micrographia and was the first person
to observe and accurately describe microorganisms (bacteria and protozoa) called
‘animalcules’ (little animals) in 1676.
 Micrographia is important for the information it provided on building
microscopes. One design discussed in Micrographia was probably a prototype
for the microscopes built and used by the amateur microscopist Antony van
Leeuwenhoek (1632–1723) of Delft, the Netherlands.
 Leeuwenhoek earned his living selling men’s clothing and accessories but spent
much of his spare time constructing simple microscopes composed of double
convex glass lenses held between two silver plates.
 His microscopes could magnify about 50 to 300 times, and he may have
illuminated his liquid specimens by placing them between two pieces of glass and
shining light on them at a 45-degree angle to the specimen plane. Leeuwenhoek
was the first person to produce precise and correct descriptions of bacteria and
protozoa using a microscope he made himself.
 Because of this extraordinary contribution to microbiology, Antonie van
Leeuwenhoek is considered as the “Father of microbiology” and also
considered to be the father of bacteriology and protozoology (protistology).
He wrote over 200 letters which were transmitted as a series of letters from 1674-
1723 to Royal Society in London during a 50 years period.
 This would have provided a form of dark-field illumination whereby organisms
appeared as bright objects against a dark background.

 Culture-based Methods for Studying Microorganisms

 As important as Leeuwenhoek’s observations were, the development of

microbiology essentially languished for the next 200 years until techniques for
isolating and culturing microbes in the laboratory were formulated.
 During this time, scientists grappled with subsequent studies on the role played
by microorganisms in causing disease ultimately led to what is now called the
golden age of microbiology.

 Spontaneous Generation theory

 From earliest times, people had believed in spontaneous generation—that living

organisms could develop from nonliving matter.
 This view was challenged by the Italian physician Francesco Redi (1626–1697),
who carried out a series of experiments on decaying meat, which was thought to
produce maggots spontaneously.
 Using covered and uncovered containers of meat, Redi clearly demonstrated that
maggots on decaying meat resulted from the presence of fly eggs, and meat did
not spontaneously generate maggots.

 However, Leeuwenhoek’s communications on microorganisms renewed the

controversy. Some proposed that microbes arose by spontaneous generation but

larger organisms did not. They pointed out that boiled extracts of hay or meat
gave rise to microorganisms after sitting for a while.
 In 1748, the English priest John Needham (1713–1781) suggested that the
organic matter in these extracts contained a “vital force” that could confer the
properties of life on nonliving matter.
 A few years after Needham’s experiments, the Italian priest and naturalist
Lazzaro Spallanzani (1729–1799) sealed glass flasks that contained water and
seeds and then placed the flasks in boiling water for about 45 minutes. He found
that no growth took place as long as the flasks remained sealed.
 He proposed that air carried germs to the culture medium but also commented
that external air might be required for growth of animals already in the
medium.
 The supporters of spontaneous generation responded that heating the air in sealed
flasks destroyed its ability to support life, and therefore did not discredit the
theory of spontaneous generation.
 In the mid-1800s, several investigators attempted to counter such arguments.
These experiments involved allowing air to enter a flask containing a nutrient
solution after boiling. The air was also very hot or it was filtered through sterile
cotton wool. In all cases, no microbial growth occurred in the medium. Nicolas
Appert followed the idea of Spallanzani’s work. He was a French wine maker
who showed that soups and liquids can be preserved by heating them extensively
in thick champagine bottles.
 Ignaz Semmelweis and John Snow were the two persons who showed a growing
awareness of the mode of disease transmission.
 Two German scholars Schulze (1815-1873) and Theodor Schwan (1810-
1882) viewed that air was the source of microbes and sought to prove this by-
passing air through hot glass tubes or strong chemicals into boiled infusions in
flasks. The infusion in both the cases remained free from the microbes.
 George Schroeder and Theodor Von Dusch (1854) were the first to introduce
the idea of using cotton plugs for plugging microbial culture tubes.
 Darwin (1859) in his book, ‘Origin of the Species’ showed that the human body
could be conceived as a creature susceptible to the laws of nature. He was of the
opinion that disease may be a biological phenomenon, rather than any magic.
 Despite these experiments, the French naturalist Felix Pouchet (1800–1872)
claimed in 1859 to have carried out experiments conclusively proving that
microbial growth could occur without contact with air.
 Pouchet’s claim provoked Louis Pasteur (1822–1895) to settle the matter of
spontaneous generation.
 Pasteur first filtered air through cotton and found that objects resembling plant
spores had been trapped. If a piece of the cotton was placed in sterile medium
after air had been filtered through it, microbial growth occurred.
SWAN-NECK FLASKS EXPERIMENT
 Next, he placed nutrient solutions in flasks, heated their necks in a flame, and
pulled them into a variety of curves. The swan-neck flasks he produced in this
way remained open to the atmosphere.
 Pasteur then boiled the solutions and allowed them to cool. No growth took place
even though the contents of the flasks were exposed to the air.
 Pasteur inferred that growth did not occur because dust and germs had been
trapped on the walls of the curved necks. If the necks were broken, growth
commenced immediately.
 Pasteur had not only resolved the controversy by 1861 but also had shown how to
keep solutions sterile.
 The English physicist John Tyndall (1820–1893) and the German botanist

Ferdinand Cohn (1828–1898) dealt the final blow to spontaneous generation.

 In 1877 Tyndall demonstrated that dust did indeed carry germs and that if dust
was absent, broth remained sterile even if directly exposed to air.
 During the course of his studies, Tyndall provided evidence for the existence of
exceptionally heat-resistant forms of bacteria and concluded that some
microorganism exists in two forms that is - a heat labile form (Vegetative) &
heat-resistant form (endospores). He developed a method of sterilization by
discontinuous heating, called Tyndallisation. Which could be used to kill all
bacteria in infusions.
 Working independently, Cohn discovered that the heat-resistant bacteria
recognized by Tyndall were species capable of producing bacterial endospores.
 Cohn later played an instrumental role in establishing a classification system for
bacteria based on their morphology and physiology.
 These early microbiologists disproved spontaneous generation thereby
contributing to the rebirth of microbiology.
 They developed liquid media and the methods for sterilizing it so that microbes
could be cultured.
 These techniques were next applied to understanding the role of microorganisms
in disease.

 Microorganisms and Disease

 For hundreds of years, most people believed that disease was caused by
supernatural forces, poisonous vapors, and imbalances among the four humors
thought to be present in the body. The role of the four humors (blood, phlegm,
yellow bile [choler], and black bile [melancholy]) in disease had been widely
accepted since the time of the Greek physician Galen (129–199).
 Support for the idea that microorganisms cause disease—that is, the germ theory
of disease—began to accumulate in the early nineteenth century from diverse
fields. Agostino Bassi (1773–1856) demonstrated in 1835 that a silkworm
disease was due to a fungal infection.
 In 1845 M. J. Berkeley (1803–1889) proved that the great potato blight of
Ireland was caused by a protozoan (then thought to be a fungus), and in 1853
Heinrich de Bary (1831–1888) showed that fungi caused crop diseases.

 Fermentations
 Pasteur was trained as a chemist and spent many years studying the fermentations
that yield ethanol and are used in the production of wine and other alcoholic
beverages.
 When he began his work, leading chemists were convinced that fermentation was
due to a chemical instability in sugars that resulted in their breakdown into
alcohol. Pasteur did not agree; he believed that fermentations were carried out by
living organisms.
 In 1856 M. Bigo, an industrialist in Lille, France, where Pasteur worked,
requested Pasteur’s assistance. His business produced ethanol from the
fermentation of beet sugars, and the alcohol yields had recently declined and the
product had become sour.
 Pasteur discovered that the fermentation was failing because the yeast normally
responsible for alcohol formation had been replaced by bacteria that
produced acid rather than ethanol.
 In solving this practical problem, Pasteur demonstrated that fermentations were
due to the activities of yeasts and bacteria.
 For several years, poor-quality wines had been produced. Pasteur referred to the
wines as diseased and demonstrated that particular wine diseases were linked to
particular microbes contaminating the wine.

Cagniard-Latour Schwann Kützing

 He eventually suggested a method for heating the wines to destroy the

undesirable microbes. The process is now called pasteurization.
 Cagniard-Latour, Schwann, and Kützing independently discovered that yeast is
responsible for the fermentation of sugar to alcohol:
 In 1837, Cagniard-Latour concluded that yeast is a living organism that breaks
down sugar and converts it to alcohol. He also suggested that yeast might be of
interest to physiologists.
 Schwann concluded that yeast is required for fermentation and that the
conversion of sugar to alcohol is a biological process.
 Kützing published drawings and descriptions of yeast cells in 1837. He estimated
the diameter of the cells to be around 6–9 μm.
 Indirect evidence for the germ theory of disease came from the work of the
English surgeon Joseph Lister (1827–1912) on the prevention of wound
infections. Lister, impressed with Pasteur’s studies on fermentation, developed a
system of antiseptic surgery designed to prevent microorganisms from entering
wounds.
 Instruments were heat sterilized, and phenol was used on surgical dressings and at
times sprayed over the surgical area. Lister concluded that wound infections too
were due to microorganisms.
 In 1867, he developed a system of antiseptic surgery designed to prevent
microorganisms from entering wounds by the application of phenol on surgical
dressings and at times it was sprayed over the surgical areas. He also devised a
method to destroy microorganisms in the operation theatre by spraying a fine mist
of carbolic acid into the air, thus producing an antiseptic environment.
 Thus, Joseph Lister was the first to introduce aseptic techniques for control of
microbes by the use of physical and chemical agents which are still in use today.
Because of this notable contribution, Joseph Lister is known as the Father of
Antiseptic surgery.
 The approach was remarkably successful in reducing the rate of surgical
infection. It also provided strong indirect evidence for the role of microorganisms
in disease.

 Koch’s Postulates

 The first direct demonstration that bacteria cause disease came from the study of
anthrax by the German physician Robert Koch (pronounced “Koke”; 1843–
1910). Koch used the criteria proposed by his former teacher Jacob Henle
(1809–1885) and others to establish the relationship between Bacillus anthracis
and anthrax.
 In these studies, published in 1876, Koch used mice as his model or test
organism, he next used guinea pigs to show that Mycobacterium tuberculosis
causes tuberculosis (TB), which at that time was a leading cause of death in
Europe.
 In 1905 Koch was awarded the Nobel Prize in Physiology or Medicine and his
criteria for proving the causal relationship between a microorganism and a
specific disease are known as Koch’s postulates.
 Koch’s postulates have since been used to discover the causative microorganisms
for many infectious diseases.
 While Koch’s postulates are still widely used, their application is at times not
feasible. For instance, viruses and organisms that must live within host cells such
as Mycobacterium leprae, the causative agent of leprosy, cannot be isolated in
pure culture. Some human diseases lack an appropriate animal model so the
postulates cannot be fully met.
 To avoid some of these difficulties, microbiologists sometimes use molecular and
genetic evidence. For instance, molecular methods might be used to detect the
nucleic acid of a microorganism in body tissues, rather than isolating it, or the
genes thought to be associated with the virulence of a disease-causing microbe
(pathogen) might be mutated. In this case, the mutant organism should have
decreased ability to cause disease.
 Introduction of the normal gene back into the mutant should restore the
pathogen’s virulence. Our focus thus far has been on the discovery of bacteria,
fungi, and protists. But viral pathogens were also being studied during this time.
 The discovery of viruses and their role in disease was made possible when
Charles Chamberland (1851–1908), one of Pasteur’s associates, constructed a
porcelain filter to remove bacteria.
 Dimitri Ivanowski (1864–1920) and Martinus Beijerinck (1851–1931) used the
filter to study tobacco mosaic disease.
 They found that plant extracts and sap from diseased plants were infectious, even
after being filtered with Chamberland’s filter.
 Because the infectious agent passed through a filter that trapped bacterial cells,
they reasoned that the agent must be something smaller than a bacterium.
 Beijerinck proposed that the agent was a “filterable virus” (Latin virus, slimy
liquid, poison). Eventually viruses were shown to be tiny, acellular infectious
agents.
 Immunology

 The ability to culture microbes also played an important role in early

immunological studies.
 During studies on the bacterium that causes chicken cholera, Pasteur and Pierre
Roux (1853–1933) discovered that bacteria incubated in cultures for long periods
of time lost their ability to cause disease.
 These bacteria were said to be attenuated or weakened.
 When chickens were injected with attenuated bacteria, they remained healthy and
were surprisingly able to resist the disease when exposed to virulent strains of the
same bacteria.
 Pasteur called the attenuated bacteria a vaccine (Latin vacca, cow) in honor of
Edward Jenner (1749–1823) because, many years earlier, Jenner had used
material from cowpox lesions to protect people against smallpox.
 Shortly after this, Pasteur and Chamberland developed an attenuated anthrax
vaccine, immunize susceptible populations.
 Pasteur also prepared a vaccine using an attenuated strain of rabies virus.
 The early advances in immunology were made without understanding how the
immune system works.
 Immunologists now know that white blood cells and the chemicals they produce
play a central role in immunity. Among the chemicals are soluble proteins called
antibodies, found in blood, lymph, and other body fluids.
 The role of antibodies in preventing disease was recognized by Emil von
Behring (1854–1917) and Shibasaburo Kitasato (1852–1931). After the
discovery that diphtheria was caused by a toxin produced by bacteria, they
injected inactivated diphtheria toxin into rabbits.
 The inactivated toxin induced rabbits to produce an antitoxin, which protected
against the disease. Antitoxins are now known to be antibodies that specifically
bind and neutralize toxins.
 The first immune system cells were discovered when Elie Metchnikoff (1845–
1916) found that some white blood cells could engulf disease-causing bacteria.
He called these cells phagocytes and the process phagocytosis (Greek phage in,
eating).
 Microbial Ecology

 Early microbial ecologists studied microbial involvement in the carbon, nitrogen,

and sulfur cycles.
 The Russian microbiologist Sergei Winogradsky (1856–1953) made many
contributions to soil microbiology, including the discovery that soil bacteria could
oxidize iron, sulfur, and ammonia to obtain energy and that many of these
bacteria could incorporate CO2 into organic matter much as photosynthetic
organisms do. Winogradsky also isolated anaerobic nitrogen-fixing soil
bacteria and studied the decomposition of cellulose.
 Martinus Beijerinck made fundamental contributions to microbial ecology as
well as virology. He isolated several kinds of nitrogen-fixing bacteria and sulfate-
reducing bacteria.
 Beijerinck and Winogradsky also developed enrichment culture techniques and
selective media, which have been of great importance in microbiology;
Biogeochemical cycling sustains life on Earth.

MICROBIAL PHYLOGENY

 Phylogenetic or phyletic classification systems compare organisms on the basis

of evolutionary relationships.
 The term phylogeny (Greek phylon, tribe or race; genesis, generation or origin)
refers to the evolutionary development of organisms.
 As discussed, microbial phylogeny relies on comparisons of multiple features
found in extant organisms.
 These include cell wall structure, biomolecules such as fatty acids, and certain
housekeeping proteins (proteins used to maintain cellular life, therefore found in
many different organisms), and nucleotide sequences, particularly of small
subunit rRNA molecules (SSU rRNA).

 Phylogenetic Trees

 The goal of phylogenetic tree construction is to display the evolutionary

relationships between different organisms.
 Trees are built by comparing the sequences of amino acids or nucleotides from
diverse organisms.
 Amino acid sequences are often compared because nucleotide changes may not
alter the resulting protein and therefore have little or no impact on evolution.
 Historically, the rRNAs from small ribosomal subunits (16S from bacteria and
archaea and 18S from eukaryotes) were the molecules of choice for inferring
microbial phylogenies and making taxonomic assignments for several reasons.
 Like housekeeping proteins (proteins with essential functions), SSU rRNAs play
the same role in all microorganisms and are absolutely necessary for survival.
 Therefore neither housekeeping proteins nor genes encoding SSU rRNAs tolerate
large changes in sequence.
 The utility of SSU rRNAs is extended by the presence of certain sequences within
SSU rRNA genes that vary among organisms as well as other regions that are
quite similar.
 The variable regions enable comparison between closely related microbes,
whereas the stable sequences allow the comparison of more distantly related
microorganisms.
 The stability of SSU rRNA sequences means that they evolve very slowly, so
analysis of housekeeping genes is more frequently used to infer the evolution of
more closely related organisms.
 Comparative analysis of SSU rRNA sequences from thousands of organisms has
demonstrated the presence of oligonucleotide signature sequences.
 These are short, conserved nucleotide sequences specific for phylogenetically
defined groups of organisms. Thus the signature sequences found in bacterial
rRNAs are rarely or never found in archaeal rRNAs, and vice versa.
 Likewise, the 18S rRNA of eukaryotes bears signature sequences that are
specific to the domain Eukarya.

 Approaches to building a phylogenetic tree can be divided into two broad

categories:

 Distance-based approaches are the most intuitive.

 This value serves as a measure of the evolutionary distance between the
organisms; the more differences counted,the greater the evolutionary distance.
 The evolutionary distances from many comparisons are used by sophisticated
computer programs to construct the tree.
 The tip of each branch in the tree (called a node) represents one of the organisms
used in the comparison.
 The distance from one node to another is the evolutionary distance between the
two organisms.
 An algorithm analyzes this information to generate a tree.
 One algorithm called cluster analysis serially links pairs that are ever more
distantly related (i.e., start with those with the least number of sequence
differences and move to those with the most).
 Although this is the easiest to understand, it can generate trees based on artifact.
Neighbor joining is another distance-based method that uses a different matrix to
modify the distance between each pair of nodes based on the average divergence
from all other nodes.
 Character-based approaches for phylogenetic tree building are more
complicated but generate more robust trees.
 These methods start with assumptions about the pathway of evolution, infer the
ancestor at each node, and choose the best tree according to a specific model of
evolutionary change.
 Sometimes called tree-searching, these methods include maximum parsimony,
which assumes that the fewest number of changes occurred between ancestor and
extant organisms.
 Another approach is called maximum likelihood. This requires a large data set,
because for each possible tree that can be built, its probability (i.e., the likelihood)
based on certain evolutionary and molecular information is determined so that the
tree with the greatest probability based on these criteria is selected.
 Bayesian inference is another character-based approach. Rather than looking at a
single tree, Bayesian inference analyzes multiple potential trees and calculates the
probability that each branch would appear based on this comparison.
 Once a tree is constructed, it is important to get a sense of whether the placement
of its branches and nodes is legitimate.
 There are a variety of methods to assess the strength of a tree, but the most
common is bootstrapping.
 Bootstrapping involves reanalyzing a randomly selected subset of the data
presented on the tree. A bootstrap value is the percent of times in which that
particular branch was found.
 Typically, bootstrap values of 70% or greater are thought to support a tree. Of
note is that Bayesian inference values are also reported as percentages, but they
are not directly comparable to bootstrap values. Only values greater than 95% are
acceptable when Bayesian inference is used.
 Two things should be kept in mind when examining phylogenetic trees. The first
is that they are molecular trees, not organismal trees. In other words, they
represent, as accurately as possible, the evolutionary history of a molecule (e.g.,
rRNA).
 Second, the distance between nodes is a measure of relatedness, not of time. If
the distance along the lines is very long, then the two organisms are more
evolutionarily diverged (i.e., less related).
 Importantly, a tree may be unrooted or rooted. An unrooted tree represents
phylogenetic relationships but does not indicate which organisms are more
primitive relative to the others.
 In contrast, the rooted tree includes a node (taxonomic unit) that serves as the
common ancestor and shows the development of the four species from this root.
It is much more difficult to develop a rooted tree. For example, there are 15
possible rooted trees that connect four species but only three possible unrooted
trees.
 An unrooted tree can be rooted by adding data from an outgroup—a species
known to be very distantly related to all the species in the tree.
 The root is determined by the point of the tree where the outgroup joins. This
provides a point of reference to identify the oldest node on the tree, which is the
node closest to the outgroup.

 Microbial Taxonomy

 The science of classifying living things is called taxonomy. Taxonomy consists

of three separate but interrelated parts: classification, nomenclature (naming),
and identification.
 A taxonomic scheme is used to arrange organisms into groups called taxa (s.,
taxon) based on mutual similarity.
 Microbes are placed in taxonomic levels arranged in a non-overlapping hierarchy
so that each level includes not only the traits that define the rank above it but also
a new set of more restrictive traits.
 Thus, within each domain—Bacteria, Archaea, or Eukarya—each organism is
assigned (in descending order) to a phylum, class, order, family, genus, and
species epithet or name. Some microbes are also given a subspecies designation.
 Microbial groups at each level have a specific suffix that indicates rank or level.
 Microbiologists name microbes using the binomial system of the eighteenth-
century biologist and physician Carl Linnaeus.
 The Latin, italicized name consists of two parts. The first part, which is
capitalized, is the generic name (i.e., the name of the genus to which the microbe
belongs), and the second is the uncapitalized species epithet. For example, the
bacterium that causes plague is called Yersinia pestis.
 Often the name of an organism will be shortened by abbreviating the genus name
with a single uppercase letter (e.g., Y. pestis).
 This straightforward organizational approach is complicated by the fact that
bacteria and archaea do not reproduce sexually.
 This definition also is appropriate for the many eukaryotic microbes that
reproduce sexually.
 However, bacterial and archaeal species cannot be defined by this criterion
because they do not reproduce sexually. Therefore, comparisons of genome
sequences are often used to distinguish one species from another.
 A common definition is that bacterial and archaeal species are a collection of
strains that share many stable properties and differ significantly from other
groups of strains. A strain consists of the descendants of a single, pure microbial
culture. Strains within a species may be described in a number of different ways.
 Biovars are variant strains characterized by biochemical or physiological
differences, morphovars differ morphologically, serovars have distinctive
properties that can be detected by antibodies, and pathovars are pathogenic strains
distinguished by the plants in which they cause disease.
 Although microbiologists continue to use Linnaeus’s classification system, the
ongoing explosion in metagenomic analysis has had an impact on this historically
accepted hierarchy.
 Within the last few decades, thousands of 16S rRNA genes and protein-coding
genes have been sequenced that do not belong to any previously defined taxa.
 These data have led to the recent development of the taxonomic classification
superphylum, below domain and above phylum.
 A superphylum includes organisms of several phyla that share a number of
distinctive characteristics, such as unusual morphological or metabolic features.

APPLIED AREAS OF MICROBIOLOGY IMPORTANCE OF

MICROBIOLOGY

 Microbiology today is as diverse as the organisms it studies. It has both basic and
applied aspects.
 The basic aspects are concerned with the biology of microorganisms themselves.
 The applied aspects are concerned with practical problems such as disease, water
and wastewater treatment, food spoilage and food production, and industrial uses
of microbes.
 Despite this apparent dichotomy, the basic and applied aspects of microbiology
are intertwined. Basic research is often conducted in applied fields, and
applications often arise out of basic research.
 An important development in microbiology is the increasing use of molecular and
genomic methods to study microbes and their interactions with other organisms.
These methods have led to a time of rapid advancement that rivals the golden age
of microbiology.

 MAJOR FIELDS IN MICROBIOLOGY

 Microbiology is commonly divided into sub-disciplines based on the type of

microbe studied. Thus, microbiology encompasses bacteriology and virology as
well as other microbe-specific fields.
 Microbiology can also be divided based on the activities of microbes—for
instance, environmental microbiology and agricultural microbiology.
 Finally, microbiologists may study only one aspect of the biology of microbes,
leading to sub-disciplines such as microbial genetics and microbial physiology.

 MEDICAL MICROBIOLOGY

 One of the most active and important fields in microbiology is medical

microbiology, which deals with diseases of humans. Medical microbiologists
investigate agents causing infectious diseases and measures for their control and
elimination. They are involved in tracking down new, unidentified pathogens
such as those causing Zika virus and COVID-19. These microbiologists also
study how microorganisms cause disease. Clinical laboratory scientists, the
microbiologists who work in hospitals and other clinical laboratories, use culture
and molecular techniques to provide information needed by physicians to
diagnose and treat infectious disease.
 Major epidemics have regularly affected human history. For example, the human
immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS) has
infected about 75 million people since the beginning of the AIDS pandemic in
1981. The COVID-19 pandemic demonstrated to the world that public health
microbiology is concerned with the control and spread of such communicable
diseases.
 Public health microbiologists and epidemiologists monitor the amount of
disease in populations to detect outbreaks and epidemics as they begin, and
implement appropriate control measures. They also conduct surveillance for new
diseases as well as bioterrorism events.
 Public health microbiologists working for local governments monitor community
food establishments and water supplies to ensure they are safe and free from
pathogens.
 Epidemiology and public health microbiology - To understand, treat, and control
infectious disease, it is important to understand how the immune system protects
the body from pathogens; this question is the concern of immunology.
 Immunology is one of the fastest growing areas in science. Many advances have
been in response to the discovery of HIV, which specifically targets cells of the
immune system.
 Immunology also deals with the nature and treatment of allergies and
autoimmune diseases such as rheumatoid arthritis.

 MICROBIAL ECOLOGY

 Microbial ecology is another important field in microbiology.

 Microbial ecologists employ a variety of culture and molecular approaches to
describe the vast diversity of microbes in terms of their morphology, physiology,
and relationships with organisms and the components of their habitats.
 The importance of microbes in the local and global cycling of carbon, nitrogen,
and sulfur has been long studied; however, these studies have acquired new
urgency as our climate changes.
 Of particular interest is the role of microbes in both the production and removal
of greenhouse gases such as carbon dioxide and methane. Microbial ecologists
and bioremediation harness microbes to clean the environment.

 AGRICULTURAL MICROBIOLOGY

 Agricultural microbiology is a field related to both medical microbiology and

microbial ecology. It is concerned with the impact of microorganisms on food
production, such as nitrogen-fixing bacteria, which affect soil fertility. Other
microbes live in the digestive tracts of ruminants such as cattle and break down
the plant materials these animals ingest. There are also plant and animal
pathogens that have significant economic impact if not controlled. Furthermore,
some pathogens of domestic animals also cause human disease.
 Agricultural microbiologists work on methods to increase soil fertility and crop
yields, study rumen microorganisms to increase meat and milk production, and
try to combat plant and animal diseases.
 Currently many agricultural microbiologists are studying the use of bacterial and
viral insect pathogens as substitutes for chemical pesticides.

 FOOD MICROBIOLOGY
 In addition to agricultural microbiology, food microbiology has contributed to the
ready supply of high-quality foods.
 Food microbiologists study the microbes used to make food and beverages (e.g.,
yogurt, cheese, beer) as well as the microbes that cause food spoilage or are
pathogens that are spread through food. For example, periodic outbreaks of
certain Escherichia coli strains have led to renal failure and death.
 To protect the public, the specific strain must be traced back to the contaminated
food source. Food microbiologists also work to prevent microbial spoilage of
food and conduct research on the use of microorganisms as nutrient sources for
livestock and humans.

 INDUSTRIAL MICROBIOLOGY

 Industrial microbiology involves the use of microbes to make products helpful to

humans. An important advance occurred in 1929 when Alexander Fleming
rediscovered that the fungus Penicillium sp. produced what he called penicillin,
the first antibiotic that could successfully control bacterial infections.
 Although it took World War II for scientists to learn how to mass-produce
penicillin, microbiologists soon found other microbes capable of producing
additional antibiotics.
 Today industrial microbiologists also use microorganisms to make products such
as vaccines, steroids, alcohols and other solvents, vitamins, amino acids,
enzymes, and biofuels.
 These alternative fuels are renewable and may help reduce fossil fuel
dependence. Biofuel production is a dynamic field; Microbial fuel cells: batteries
powered by microbes.
 The advances in medical microbiology, agricultural microbiology, food and dairy
microbiology, and industrial microbiology are outgrowths of basic research in
areas such as microbial physiology, microbial genetics, molecular biology, and
bioinformatics.
 Microbial physiologists study many aspects of the biology of microorganisms,
including their diverse metabolic capabilities. They also study the synthesis of
antibiotics and toxins, the ways in which microorganisms survive harsh
 environmental conditions, and the effects of chemical and physical agents on
microbial growth and survival.
 Microbial geneticists, molecular biologists, and bioinformaticists study the
nature of genetic information and how it regulates the development and function
of cells and organisms.
 The bacteria E. coli and Bacillus subtilis, the yeast Saccharomyces cerevisiae
(baker’s yeast), and bacterial viruses such as T4 and lambda continue to be
important model organisms used to understand biological phenomena.
 Genomics has revolutionized biology, as scientists are now beginning to
understand organisms holistically, rather than in a reductionist, piecemeal
manner.
 How the genomes of microbes evolve, the nature of host-pathogen interactions,
the minimum set of genes required for an organism to survive, and many more
topics are aggressively being examined by molecular and genomic analyses.