HISTORY OF MICROBIOLOGY: SPONTANEOUS GENERATION THEORY

Microbiology often has been defined as the study of organisms and agents too small to
be seen clearly by the unaided eye—that is, the study of microorganisms. Because objects less
than about one millimeter in diameter cannot be seen clearly and must be examined with a
microscope, microbiology is concerned primarily with organisms and agents this small and
smaller.

Microbial World
Microorganisms are everywhere. Almost every natural surface is colonized by microbes
(including our skin). Some microorganisms can live quite happily in boiling hot springs, whereas
others form complex microbial communities in frozen sea ice.
Most microorganisms are harmless to humans. You swallow millions of microbes every
day with no ill effects. In fact, we are dependent on microbes to help us digest our food and to
protect our bodies from pathogens. Microbes also keep the biosphere running by carrying out
essential functions such as decomposition of dead animals and plants.
Microbes are the dominant form of life on planet Earth. More than half the biomass on
Earth consists of microorganisms, whereas animals constitute only 15% of the mass of living
organisms on Earth.

This Microbiology course deals with

• How and where they live
• Their structure
• How they derive food and energy
• Functions of soil micro flora
• Role in nutrient transformation
• Relation with plant
• Importance in Industries

The microorganisms can be divided into two distinct groups based on the nucleus
structure:
Prokaryotes – The organism lacking true nucleus (membrane enclosed chromosome and
nucleolus) and other organelles like mitochondria, golgi body, entoplasmic reticulum etc. are
referred as Prokaryotes. (Ex : Bacteria, archaea)
Eukaryotes - The organism possessing membrane enclosed nucleus and other cell organelles
are referred as Eukaryotes (Ex : algae, fungi, protozoa)
The microorganisms were divided into 6 distinct groups based on the phylogenic, morphological
and physiological characters.

The major groups of microorganisms are

1. Bacteria are phylogenetically related group of unicellular prokaryotic organisms distinct
from archeae
2. Archaea is phylogenetically related group of prokaryotes which are primitive and distinct
from bacteria
3. Fungi are group of eukaryotic organisms lacking chlorophyll. They range in size and
shape from single celled yeast to multicellular mushrooms.
4. Algae refer the group of eukaryotic organisms with chlorophyll. They range in size and
shape from single celled algae (Ex: Chlorella) to complex cellular structured plant like
algae (Ex. Kelp)
5. Protozoa are group of eukaryotic organisms lack of cell wall. The morphology, nutrition
and physiology is different from other groups
6. Viruses are group of non-cellular organisms, parasite or pathogen to plant, animals and
other microorganisms. They are too small and cab be visualized only under electron
microscopes

History of Microbiology in brief

Obviously human have had to deal with microbes even before the recorded history. The
first record of human using comes from ancient tablets from mid east.
Babylonians were using yeast to make beer over 8000 years ago and acetic acid
bacteria to make vinegar over 6000 years ago.
About 5000 years ago, Persia (Now Iran) region recorded the wine making.
The Romans had God for that were specific for microorganisms. The roman God of
mold and mildew was “Robigus” and “Robigo” which means crop rust. (Rust is one of the plant
disease caused by fungus). God Robigus was very much feared because of crop lost.
About 2000 years ago, Romans proposed that diseases were caused by tiny animals.
But, fundamentalist religions had a strong hold over the progress.
The real microbiology history starts from 1600s, when people began to make crude
lenses and microscopes.
HIGHLIGHTS IN THE HISTORY OF MICROBIOLOGY
Effects of Disease on Civilization
• Infectious diseases have played major roles in shaping human history
• Bubonic Plague epidemic of mid 1300's, the "Great Plague", reduced population of western
Europe by 25%. Plague bacterium was carried by fleas, spread from China via trade
routes and poor hygiene. As fleas became established in rat populations in Western
Europe, disease became major crisis.
• Smallpox and other infectious diseases introduced by European explorers to the Americas in
1500's were responsible for decimating Native American populations. Example: In the
century after Hernan Cortez's arrival in Mexico, the Aztec population declined from about
20 million to about 1.6 million, mainly because of disease.
• Infectious diseases have killed more
soldiers than battles in all wars
up to WW II. Example: in U. S.
Civil war, 93,000 Union
soldiers died in direct combat;
210,000 died as a result of
infections.
• Until late 1800's, no one had proved
that infectious diseases were
caused by specific microbes,
so the possibility of prevention Brueghel: The Triumph of Death (1560)
or treatment had no sound  
empirical base.

Discovery of Microbes
• To see microbes, you need a microscope. The first
microscope was invented by Antony van
Leeuwenhoek (1632-1723), a Dutch businessman.
• Leeuwenhoek took up lens grinding to make magnifiying
glasses so he could examine fine weave of fabrics.
In testing his lenses, he discovered many small

Antony  van  Leeuwenhoek  

 creatures he called "animalcules" in samples such as pond water. His best lenses could
magnify 300-500X.
• Leeuwenhoek microscopes were crude, relied on a single lens held in a metal plate.
• Leeuwenhoek described many previously unseen life forms, including different forms of
bacteria, mold spores, etc. Leeuwenhoek reported discoveries to Royal Society from
1670's on, firmly established existence of microbes. Nevertheless, the significance of
this discovery was not apparent for almost 200 years.

Origin of Life Controversy

• Where did microbes come from? Many believed they arose from simple materials by process
of spontaneous generation. This notion had been posited by Aristotle (382-322 B.C.) and
other Greek philosophers to explain decay and appearance of animals such as flies and
frogs, and was widely held as common sense even in 1700's and 1800's.

• Francisco Redi (1626-1697) demonstrated that flies did not arise spontaneously from rotting
meat by simple experiment. If jar of meat was covered by fine muslin, maggots did not
arise. However, the simpler life forms discovered by Leeuwenhoek lacked visible
complexity, and most people still believed these could arise spontaneously.

• John Needham (1731-1781), a Scottish clergyman and naturalist,

showed that mirobes grew in soups exposed to air. Claimed
existence of a "life force" present in inorganic matter that could
cause spontaneous generation. One of his more convincing
demonstrations was to boil some soup (briefly), pour into clean
flasks with cork lids, and show that microbes would soon arise.
• Lazzaro Spallanzani (1729-1799) claimed Needham's organisms
came from heat-resistant microbes. If flasks were boiled long
enough (1-2 h), nothing grew. But Needham countered that
prolonged heating destroyed the "life force".

• Louis Pasteur (1822-1895) was passionate believer that life only originated from previous
life, developed several experiments that finally deflated claims for spontaneous
generation. Pasteur filtered air through cotton to
trap airborne materials, then dissolved the cotton
and examined the particulate matter under a
microscope; many bacteria and spores of other
life forms such as molds were present. Since
most skeptics kept arguing that overheating killed
the life force present in air, Pasteur developed
and ingenious experiment using a swan neck
flask that allowed fresh air to remain in contact
with boiled materials. The long passageway
prevented airborne microbes from reaching the Louis Pasteur
 
nutrient liquid, without impeding access to air. One of Pasteur's flasks is still sterile after
100+ years of being exposed to the air (Pasteur Institute, Paris).

Spontaneous Generation theory

From earliest times, people had believed in spontaneous generation—that living
organisms could develop from nonliving matter. Even the great Aristotle (384–322 B.C.) thought
some of the simpler invertebrates could arise by spontaneous generation. This view finally was
challenged by the Italian physician Francesco Redi (1626–1697), who carried out a series of
experiments on decaying meat and its ability to produce maggots spontaneously. Redi placed
meat in three containers. One was uncovered, a second was covered with paper, and the third
was covered with a fine gauze that would exclude flies. Flies laid their eggs on the uncovered
meat and maggots developed. The other two pieces of meat did not produce maggots
spontaneously. However, flies were attracted to the gauze-covered container and laid their eggs
on the gauze; these eggs produced maggots. Thus the generation of maggots by decaying
meat resulted from the presence of fly eggs, and meat did not spontaneously generate maggots
as previously believed. Similar experiments by others helped discredit the theory for larger
organisms.
Leeuwenhoek’s discovery of microorganisms renewed the controversy. Some proposed
that microorganisms arose by spontaneous generation even though larger organisms did not.
They pointed out that boiled extracts of hay or meat would give rise to microorganisms after
sitting for a while. In 1748 the English priest John Needham (1713–1781) reported the results of
his experiments on spontaneous generation. Needham boiled mutton broth and then tightly
stoppered the flasks. Eventually many of the flasks became cloudy and contained
microorganisms. He thought organic matter contained a vital force that could confer the
properties of life on nonliving matter. A few years later the Italian priest and naturalist Lazzaro
Spallanzani (1729–1799) improved on Needham’s experimental design by first sealing glass
flasks that contained water and seeds. If the sealed flasks were placed in boiling water for 3/4 of
an hour, 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 the external air might be required
for growth of animals already in the medium. The supporters of spontaneous generation
maintained that heating the air in sealed flasks destroyed its ability to support life. Several
investigators attempted to counter such arguments. Theodore Schwann (1810–1882) allowed
air to enter a flask containing a sterile nutrient solution after the air had passed through a red-
hot tube. The flask remained sterile. Subsequently Georg Friedrich Schroder and Theodor von
Dusch allowed air to enter a flask of heat-sterilized medium after it had passed through sterile
cotton wool. No growth occurred in the medium even though the air had not been heated.
Despite these experiments the French naturalist Felix Pouchet claimed in 1859 to have carried
out experiments conclusively proving that microbial growth could occur without air
contamination.
This claim provoked Louis Pasteur (1822–1895) to settle the matter once and for all.
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 appeared. Next he placed nutrient solutions in flasks, heated their necks in a
flame, and drew them out into a variety of curves, while keeping the ends of the necks open to
the atmosphere .Pasteur then boiled the solutions for a few minutes and allowed them to cool.
No growth took place even though the contents of the flasks were exposed to the air. Pasteur
pointed out that no growth occurred 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) dealt a final blow to spontaneous generation in
1877 by demonstrating 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. Working
independently, the German botanist Ferdinand Cohn (1828–1898) discovered the existence of
heat-resistant bacterial endospores

The Spontaneous Generation Experiment: Pasteur’s swan neck flasks used in his
experiments on the spontaneous generation of microorganisms.

2. Disproval of Spontaneous Generation theory

At that time, the age old idea of “Spontaneous Generation theory” was the dominant
one. The idea that organism originate directly from non-living matter. (Life from non-living) also
called as abiogenesis (a – not; bio – life; genesis – origin).
Eg : Maggots were developed spontaneously via recombination of matters in rotting materials.
(ex meat)
Microbiology starts with the disproval of SG theory.
Louis Pasteur (1822 – 1895) and disproval of Spontaneous generation theory
He performed “gooseneck experiment”. The nutrient of flask was heated and the
untreated – unfiltered air could pass in or out, but the germs settled in the gooseneck and no
microbes were observed in the nutrient solution.
His concept of Germs theory of disease (means germs are responsible for the disease
not the inert mater) ends the SG theory.

Contributions of Louis Pasteur (1822 – 1895)

• Disproved the SG theory
• Discovered that fermenting fruit to alcohol by microbes – From now the Fermentation
started
• Sorted different microbes giving different taste of wine.
• He selected a particular strain (Yeast) for high quality wine.
• He developed a method to remove the undesired microbes from juice without affecting
its quality. Heating the juice at 62.8°C for half-an hour did the job. This technique is
called as Pasteurization, which is commonly used in the field of milk industry.
• He discovered that parasites (protozoa) causing pebrine disease of silk worm. He
suggested that disease free caterpillars can eliminate the disease.
• He isolated the anthrax causing bacilli from the bloods of cattle, sheep and human
being.
• He also demonstrated the virulence (ability of microbe to cause disease) of bacteria
• He developed vaccine (a killed or attenuated microbe to induce the immunity) against
rabbis from the brains and spinal cord of rabbit

John Tyndall (1820 -1893)

• Proved that dust carries the germs and if no dust in the air, the sterile broth remained
free of microbial growth for indefinite period.
• He also developed a sterilization method “Tyndallization”, referred as intermittent or
fractional sterilization. The subsequent cooling and heating by steam for 3 days will
remove the germs and their spores.

Martinus Willium Beijerinck (1851 – 1931)

• Developed the enrichment technique to isolate various group of bacteria.
• Isolated sulphur reducing bacteria and sulphur oxidizing bacteria from soil
 • Isolated free-living nitrogen fixing bacterium, Azotobacter
from soil,
• Root nodulating bacterium, Rhizobium, Lactobacillus,
green algae were identified by him
• He confirmed the Tobacco mosaic virus causes disease
and it incorporated in the host plant to reproduce.

Sergei Winogradsky (1856 – 1953)

The following are the contributions of Winogradsky to soil
microbiology.
• Microorganisms involved in N cycle, C cycle, S cycle
• Nitrification process in soil
• Autotrophic nutrition of bacteria
• Chemolithotrophic nutrition of soil bacteria
• Discovered anaerobic nitrogen fixing bacterium
Clostridium pasteurianum

Walther Hesse & Fannie E. Hesse (1883)

They used agar instead of gelatin for preparation of
media. Agar goes to solution at 100°C and solidifies at 45°C.
Till now this was not replaced by any other substance.
Joseph Lister (1878)
Developed Pure culture technique. Pure culture
referred as the growth of mass of cells of same species in a
vessel. He developed the pure cultures of bacteria using serial
dilution technique.
 He also discovered that carbolic acid to disinfect the surgical equipments and dressings
leads the reduction of post-operational deaths/infections.

Alexander Fleming (1928) identified Penicillium notatum inhibiting Staphylococcus aureus and
identified the antibiotic Penicillin
• 1929-Discovered antibiotic
penicillin –important
milestone in medical
microbiology
• Found that natural
substances having
antimicrobial activity-
Saliva,Nasal mucous
• Worked on Staphylococcus
aureus,-inhibition of growth-
due to Penicillin
• Florey &Chain-isolated Penicillin in pure culture.

Selman A Waksman, 1945 identified Streptomycin antibiotic from soil bacterium. He also
coined the term antibiotics (referring a chemical substance of microbial origin which is in small
quantity exert antimicrobial activity.
• 1927- Wrote the book on Principles of soil Microbiology
• In 1939 Waksman and his colleagues undertook a
systematic effort to identify soil organisms producing
soluble substances that might be useful in the control of
infectious diseases, what are now known as antibiotics
• Within a decade ten antibiotics were isolated and
characterized,
• three of them with important clinical applications
• actinomycin in 1940, streptomycin in 1944, and
neomycin in 1949.
• Eighteen antibiotics were discovered under his general direction.
 GERM THEORY OF DISEASE
Introduction

Bacteria are mostly unicellular organisms that lack chlorophyll and are among the
smallest living things on earth—only viruses are smaller. Multiplying rapidly under
favorable conditions, bacteria can aggregate into colonies of millions or even billions of
organisms within a space as small as a drop of water.The Dutch merchant and amateur
scientist Anton van Leeuwenhoek was the first to observe bacteria and other
microorganisms. Using single-lens microscopes of his own design, he described
bacteria and other microorganisms (calling them "animacules") in a series of letters to
the Royal Society of London between 1674 and 1723.

Bacteria are classified as prokaryotes. Broadly, this taxonomic ranking reflects

the fact that the genetic material of bacteria is contained in a single, circular chain of
deoxyribonucleic acid (DNA) that is not enclosed within a nuclear membrane. The word
prokaryote is derived from Greek meaning "prenucleus." Moreover, the DNA of
prokaryotes is not associated with the special chromosome proteins called histones,
which are found in higher organisms. In addition, prokaryotic cells lack other membrane-
bounded organelles, such as mitochondria. Prokaryotes belong to the kingdom Monera.
Some scientists have proposed splitting this designation into the kingdoms Eubacteria
and Archaebacteria. Eubacteria, or true bacteria, consist of more common species,
while Archaebacteria (with the prefix archae—meaning ancient) represent strange
bacteria that inhabit very hostile environments. Scientists believe these bacteria are
most closely related to the bacteria which lived when the earth was very young.
Examples of archaebacteria are those bacteria which currently live in extremely salty
environments or extremely hot environments, like geothermal vents of the ocean floor

Microbes are organisms that we need a microscope to see. The lower limit of our
eye's resolution is about 0.1 to 0.2 mm or 100 - 200 um. Most microbes range in size
from about 0.2 um to the 200 um upper limit, although some fruiting bodies of fungi can
become much larger. Microbes include the bacteria, algae, fungi, and protozoa. In this
lecture we will discuss mostly the bacteria and the fungi.
 Bacteria are found everywhere in water, soil, and even air. These small
prokaryotic cells, typically from 0.2 to 1 um in length, are capable of living in boiling
water, frozen ground, acid volcanoes, and at the bottom of the ocean. They can
reproduce by doubling with a generation time of 20 minutes, or survive for centuries in a
resting stage. In natural waters (lakes, streams, oceans) their generation time is around
1 day. In soils they live in a film of water around plant roots or other particles, and their
activity is dependent on the temperature and the amount of available moisture. In
general, bacteria are found in concentrations of 106 cells/mL of water in surface waters,
and 109 cells/mL of soil in soils and sediments.

Robert Koch (1843 -1910): The Father of Microbial Techniques

Robert Koch, a German Physician, is well known to the world of
microbiology for this significant contributions especially in the area of
microbial techniques. He introduced analine dyes for staining bacteria; used
agar-agar and gelatin to prepare solid culture media; stressed the need for
pure culture to study microbes in details; confirmed germ theory of
disease, and laid down Koch's
postulates to test the pathogenesity of
causative agents. He also discovered
the casual organisms of anthrax
disease of cattle (Bacillus anthracis)
and tuberculosis (Mycobacterium
tuberculosis).
Robert Koch was particularly
concerned with this problem and, at
first, he cultured bacteria on solid fruits and vegetables such as slices of
boiled potato but many bacteria did not grow on such substrates. Then he
perceived that it would be far better if a well-tried liquid medium could be
solidified with some clear substance. Koch (1881) tried gelatin as a
solidifying agent and succeeded in developing solid culture media, but
gelatin, the first solidifying agent used, had serious disadvantage of
becoming liquid above 28-30°C which is below the optimum temperature
for the growth of human disease producing bacteria.
However, Koch replaced gelatin by agar in 1883-84 on the
recommendation of F.E. Hesse, a German housewife, who had gained
experience with the characteristics of agar in the process of making jelly.
Agar is still frequently used as solidifying agent in microbiological
laboratories. The development of solid culture media to grow pure culture
was of fundamental importance and may be considered one of the Koch's
greatest contributions.
Besides developing solid culture media using gelatin and agar, Koch
also evolved methods to placed microbes on glass slides and colour them
with analine dyes (stains) so that the individual cells could be seen more
clearly under the microscope.

KOCH’S POSTULATES
1. The microorganism must be present in every case of the disease but
absent from healthy organisms.
2. The suspected microorganism must be isolated and grown in a pure
culture.
3. The same disease must result when the isolated microorganism is
inoculated into a healthy host.
4. The same microorganism must be isolated again from the diseased host.

"One microbe, one disease"

• Robert Koch (1843-1910) was the first to rigorously demonstrate that a specific
disease was caused by a specific microorganism.
• Koch worked on anthrax, a disease mainly of animals. Koch noticed that cattle that
died of anthrax all seemed to have a certain rod-shaped bacterium in blood, not found in
healthy animals. Koch was able to isolate the bacterium in pure culture, put it back into
healthy cows, and reproduce the disease.
• Koch's Postulates: a logical way to identify the microbe causing a disease
A specific microbe must be present in all disease cases
Microbe must be cultivated outside host in a pure culture
When pure culture of microbe is inoculated into healthy hosts, disease
symptoms identical to those of initial host must be reproduced
Microbe can be isolated again in pure culture from this experimentally
inoculated host.
• Initial attempts to isolate microbes used sliced potatoes or nutrient media containing
gelatin -- not ideal media. Then Fannie Hesse (wife of lab worker) suggested agar, a
gelling agent used in cooking. Agar rapidly became the standard gelling agent for
microbial isolation because it is relatively inert (only some marine microbes have
enzymes to digest agar). Agar only melts at high temperatures (100oC); once melted, it
remains liquid until about 45oC, at which point it gels.
• Koch's success at identifying anthrax with bacterium Bacillus anthracis led both Koch
and Pasteur to identify the causes of many diseases -- cholera, tuberculosis, plague,
etc. -- over the next few decades (late 1880's) -- the "Golden Age of Microbiology" (~
1870-1920). Note that many microbiologists would regard the present as a new "Golden
Age", since the development of molecular biological techniques, PCR, molecular
phylogeny, and other developments have revealed many new insights and opened a
world of new research directions and ways of understanding microbes.
 PROTECTION AGAINST INFECTIONS

The control of microbial growth is necessary in many practical situations, and

significant advances in agriculture, medicine, and food science have been made
through study of this area of microbiology. The microorganisms are ubiquitous in
nature. In order to study the nature and characteristics of a particular microbe, it is
essential to isolate it from other contaminating microorganisms. This can be
achieved by maintaining a completely sterile environment in which the microbe of
interest is selectively grown. It is necessary that not only the place you are
working with microorganisms should be free from contamination (other living
organisms) but, the media and the materials you are using to handle and grow
specific microorganisms should b e f r e e f r o m o t h e r m i c r o b i a l contaminants.
For t h i s p u r p o s e ‘sterilization’ of the place of work materials and media have to be
done.
“Control of growth” as used here means to prevent the growth of
microorganisms.   This control is affected in two basic ways: (1) by killing
microorganisms or (2) by inhibiting the growth of microorganisms. Control of growth
usually involves the use of physical or chemical agents which either kill or prevent
the growth of microorganisms. Agents which kill cells are called cidal agents; agents
which inhibit the growth of cells (without killing them) are referred to as static agents.
Thus the term bactericidal refers to killing b a c t e r i a a n d b a c t e r i o s t a t i c r e f e r s
t o i n h i b i t i n g the growth o f bacterial cells. A bactericide kills bacteria; a
fungicide kills fungi, and so on.
Sterilization is a process of complete removal or killing of all forms of
microbial life including s p o r e s f r o m a n o b j e c t , s u r f a c e , m e d i u m o r
environment without spoiling its nature.

Methods
There are various sterilization techniques available. However, several
factorsinfluence the effectiveness of sterilization process like, the
concentration of antimicrobial agents, time and temperature of exposure, size of
population, type of contaminating microbes etc.
Sterilization is brought about by a combination of physical and chemical agents
that adversely affect the microorganisms either by causing damage to the cell wall
or cell membrane or by inactivating the enzymes or by interfering with the
synthesis of nucleic acids and protein.
I. PHYSICAL AGENTS
There are different types of physical agents.

(i) Heat: The heat employed for removal of micro-organisms varied with the nature of
object and also depend on the purpose. Based on these different processes are
employed.
(a) Moist heat
It is the widely used effective means of sterilization process. In this, steam
under high pressure is employed which imparts high penetration power resulting
in the hydration of cells and coagulation of protein leading to the death of the
microorganism. Autoclave is the apparatus used for sterilization by moist heat.
The autoclave is a double-jacketed steam chamber. The chamber is equipped
with a d e v i c e f o r g e n e r a t i n g s a t u r a t e d s t e a m . It c a n b e maintained
at a particular temperature and pressure for any period of time. During operation of
autoclave the air in the
chamber is evacuated by
steam since presence of air
will reduce the temperature in
the chamber. The time
required for sterilization will
depend upon the materials to
be sterilized. Solid materials
must be heated for a longer
time (1-2 hours) while liquid media can be sterilized within 15-30 minutes. Also acidic
materials require shorter period than alkali materials. A temperature of 121°C for 15
min at a pressure of 15 lbs/ sq.inch is the sterilizing condition in the autoclave.

Advantages
Steam can penetrate through materials and sterilization is achieved by the
coagulation or denaturation of proteins and other cell constituents. Liquid m e d i a ,
s o l i d m e d ia , laboratory e q u i p m e n t s (cloth, glasswares, etc.,) can be sterilized.
The temperature and pressure is high enough to kill spores, vegetative cells and
viruses.

Disadvantages
Temperature sensitive media, animal tissue culture media, antibiotics, amino acids,
cannot be sterilized. Somet imes water may get inside incase of improper packing.
(b) Dry heat
 This process is accomplished in a hot-air oven. Hot air or dry heat is
employed for sterilization. The dry heat penetrates substances more slowly than the
moist heat. Hence, the time required for effective sterilization is long (2 to 3 hours)
and also the temperature required is too high (160°C -180°C). Microbial death
results from the oxidation of cell constituents.
Advantages
Dry heat does not corrode glassware and metal instruments as moist heat does. All
glassware’s can be sterilized.
Disadvantages
The sterilization process is slow. It is not suitable for heat sensitive materials like
many plastic and rubber items.
(c) Boiling at 100°C for 30 minutes. Kills everything except some endospores
o
(Actually, for the purposes of purifying drinking water 100 for five minutes is
probably adequate though there have been some reports that Giardia cysts can
survive this process). To kill endospores, and therefore sterilize the solution, very
long or intermittent boiling is required.
(d) Pasteurization is the use of mild heat to reduce the number of
microorganisms in a product or food. In the case of pasteurization of milk the time
and temperature depend on killing potential pathogens that are transmitted in milk,
i.e., staphylococci, streptococci, Brucella abortus and Mycobacterium tuberculosis.
For pasteurization of milk:
o
batch method (Low temperature holding): 62.8 C for 30 minutes flash
o
method (High temperature short time): 71.7 C for 15 seconds
(e) Intermittent sterilization or Tyndallization is the process of boiling the materials
o
at 100 C for 30 min. successively for three consecutive days. Destroys vegetative
cells and spores; germinated spores.
(f) Incineration burns organisms and physically destroys them. Incineration is the
complete burning of the material in to ashes. Used for needles, inoculating
wires, glassware, e t c . and o b j e c t s not destroyed in the incineration
process. This is the direct and ultimate method of destroying cells. It is achieved
by keeping the materials directly in contact with the flame of Bunsen burner as a
result all the microorganisms in the surface are destroyed completely. Inoculating
loops, needles and spreading rods are sterilized by this method.
Advantages: Immediate and quick.
Disadvantages: Cannot be used to sterilize heat labile material, material is lost by
incineration.
Recommended use of heat to control bacterial growth
Treatment Temperature Effectiveness
Vaporizes organic material on non
Incineration 5000 C flammable Surfaces but may destroy
many substances in the process

30 minutes of boiling kills microbial

Boiling 100 0C pathogens and vegetative forms of
bacteria but may not kill bacterial
endospores
Three 30-minute intervals of boiling,
Intermittent boiling 1000C followed by Periods of cooling kills
bacterial endospores.

Autoclave and 1210C for 15 min. Kills all forms of life including bacterial
pressure cooker at 15 lbs/ sq.inch endospores. The substance being
(steam under pressure sterilized must be maintained at the
pressure) effective T for the full time

For materials that must remain dry

and which are not destroyed at the
Dry heat (hot air oven) o o
between 121 C and 170 C Good for
1600 C /2 hours
glassware, metal, not plastic or rubber
items
Same as above. Note increasing T by
Dry heat (hot air oven) 1800 C /1 hour 10 degrees shortens the sterilizing
time by 50 percent

kills most vegetative bacterial cells

62.8 0C /30 min. including pathogens such as
Pasteurization streptococci, staphylococci and
(batch method) Mycobacterium tuberculosis
Effect on bacterial cells similar to
batch method; for milk, this method is
Pasteurization 71.7 0C/15 seconds more conducive to industry and has
(flash method) fewer undesirable effects on quality
or taste
(ii) Radiation
Energy transmitted through space in a variety of forms is generally called
radiation. It is also known as "cold sterilization" as only little heat is produced
during the process. The most significant of this is electromagnetic radiation. The
energy content and radiation wavelength are inversely proportional to each
 other. Radiation may be ionizing or non-ionizing.
Ionizing radiation
High-energy electron beams (Gamma, X-rays, alpha and beta particles) have
sufficient energy to cause ionization of molecules. They drive away electrons and
split the molecules into ions. Water molecules are split into hydroxyl radicals (OH-),
electrons and hydrogen ions (H+). OH- ions are highly reactive and destructive to
normal cellular compounds such as DNA and proteins. Thus ionizing radiations are
used in sterilization.
36 60
e.g. Cs, Co
Advantages: X-rays and Gamma rays have high penetrating power. Packed food
and medical equipments are sterilized by using x-rays and gamma rays.
Disadvantage: Generating and controlling X-rays for sterilization is highly
expensive.
Non-ionizing radiation
This includes ultraviolet (UV) rays. UV at a wavelength of 265 nm is most
bactericidal. Absorption of UV radiation produces chemical modification of
nucleoproteins i.e., thymine dimer formation that leads to misleading of genetic
codes. This mutation impairs the total functions of the organism, consequently
causing its death.
Advantages
It is used to maintain aseptic conditions in laminar air flow chamber, lab, hospitals,
pharmaceuticals, industries etc., and also in the sterilization of water and air.
Disadvantage:
UV radiation has very little ability to penetrate matter and hence the micro organisms on
the surface of an object are destroyed.
III) Filtration
Filtration involves the passage of liquid or gas through a screen like material that
has spores small enough to retain the micro organism of certain size. It is used to
sterilize heat sensitive substance like enzyme solutions, bacterial toxins, certain
biological media, cell extract and some sugars. Various types of filters are available in
different grades of porosity. Vacuum or pressure is required to move the solutes through
the filter.
Involves the physical removal of all cells in aliquid or gas, especially important to
sterilize solutions which would be denatured by heat (eg: antibiotics, injectable drugs,
amino acids, vitamins etc.)
Advantages:
It si the best way to reduce microbial population in solutions of heat sensitive materials
and it is sued to sterilize liquid media, vitamin solutions, hormones, growth factors,
enzymes.
Disadvantages
Pleomorphic structures like mycoplasma cannot be effectively filtered by this technique.
It is applicable to sterilize only small quantities.
Commonly used filters in micro biology
The sintered glass fliter is made of fused Jen or pyrex glass, manufactured in
such a way as to be porous, with apore size and adsorptive charge sufficient to retain
bacteria. The seitz filters are compressed asbestos discs having porosity sufficiently
small to retain bacteria. Tye chamber land filters are made of porcelain. The
mandler/berkfield filters are made of diatomaceous earth. The membrane filter is a
cellulose or nitrocellulose membrane with apore size sufficiently small (0.01mm to 10
mm) to trap and thereby remove bacterial from a liquid. The membrane filters are also
used to concentrate and trap the micro organisms in water and other liquids. HEPA
(High efficiency particulate air filters are of fibre galss filters for sterilization of air.
Low temperature
Most organisms grow very little or not at all at 0O C. Store perishable foods at low
temperatures to slow rate of growth and consequent spoilage (eg: milk). Low
temperatures are not bactericidal. Psychrotrophs, rather tah true psychrophiles are the
usual cause of food spoilage in refrigerated foods.
Dessication / Drying (removal of H2O)
Most micro organisms cannot grow at reduced water activity (aw < 0.90). Often
used to preserve foods (eg: fruits, grains etc). methods involve removal of water from
product by heat, evaporation, freeze drying, addition of salt or sugar.

Surface tension
is a property of the surface of a liquid that allows it to resist an external force. It is
revealed, for example, in floating of some objects on the surface of water, even though
they are denser than water, and in the ability of some insects (e.g. water striders) and
even reptiles (basilisk) to run on the water surface. This property is caused by cohesion
of like molecules, and is responsible for many of the behaviors of liquids.

Surface tension has the dimension of force per unit length, or of energy per unit area.
The two are equivalent—but when referring to energy per unit of area, people use the
term surface energy—which is a more general term in the sense that it applies also to
solids and not just liquids.
In materials science, surface tension is used for either surface stress or surface free
energy

Osmotic pressure – plasmolysis/ plasmotysis

Is the process in plant cells where the plasma membrane pulls away from the cell
wall due to the loss of water through osmosis. The reverse process, cytolysis, can occur
if the cell is in a hypotonic solution resulting in a higher external osmotic pressure and a
net flow of water into the cell. Through observation of plasmolysis and deplasmolysis it
is possible to determine the tonicity of the cell's environment as well as the rate solute
molecules cross the cellular membrane.
Chemical agents
Chemical that is used to kill or inhibit the growth and development of micro
organisms are called anti microbial agents. Disinfectants and antiseptics come under
anti microbial agents and are usually used on inanimate materials. The mechanism of
action is complex and non specific. It may act on lipid portion of cell membrane, oxidize
or reduce an important functional group of an enzyme, prevent certain bio synthesis or
cause extensive breakdown of DNA.
Types of microbial agents
Chemical sterilants
Chemical sterilants are chemical anti microbial agents that are usd fro
sterilization of heat sensitive substance/ materials. Normally plastic petriplates and
medical supplies such as blood transfusion sets, plastic syringes, lenses etc. could be
sterilized even in packets or bundles using ethylene oxide, formaldehyde or formalin is
O
effectively used to sterilize enclosed areas/a septic chambers at 22 C with a relative
humidity of 60 – 80 %.
Antispetics
Microbicidal agents harmless enough to be applied to the skin and mucous
membrane, should not be taken internally. Eg: mercurails, silver nitrate, iodine solution,
alcohols, detergents.
Disinfectants
Agents that kill micro organisms, but not necessary their spores, not safe for
application to living tissues, they are used on inanimate objects such as tables, floors,
utensils etc. eg: Chlorine, hypochlorites, chlorine compounds, Lysol, copper sulfate,
quaternary ammonium compounds.
Phenol
Derivative of phenol like benzyl resorcinol, o-cresol, m-cresol, etc., are used as
effective disinfectants 5% aqueous solutions of phenols are used as disinfectant. It
alters the protein structure and leads to denaturation of proteins and enzymes. Also
affects permeability of cytoplasmic membrane. They readily kill vegetative cells of
bacteria and fungi but for spores.
Alcohol
Alcohol at 70% concentration is more effective. It brings about denaturation and
coagulation of protein. Ethanol is routinely used in laboratories to surface sterilize
worktables and hands of the researcher/ experiment.
Halogens
Halogens such as hypocholrites, choramines and povidone- iodine are used to
sanitize utensils, surface sterilize in animate objects, table surfaces and other
instruments.
Heavy metals
Heavy metals such as mercuric chloride are also used for surface sterilization
purposes. Heavy metals acts as oxidizing agents and kill the micro organisms on the
surface of the object. Usually 0.1 % mercuric chloride is used in the laboratories to
sterilize the surface of worktable and explants.
Detergents
Detergents are those compounds that make water repellant surfaces more
wettable. There are two types of detergents viz., ionic and non ionic. Detergent soaps
and other synthetic detergents are used for washing/cleaning glass wares, table tops
etc.,
Common antiseptics and disinfectants
Chemical Action Uses

Ethanol (50 -70 %) Denatures proteins and Anti septic used on skin
solubilizes lipids.

Isopropanol (50 – 70 %) Denatures proteins and Anti septic used on skin.

solubilizes lipids.

Formaldehyde (8%) Reacts with NH2, SH and Disinfectant, kills

COOH groups. endopsores.

Tincture of Iodine ( 2% in Inactivates proteins Antiseptic used on skin

70 % alcohol)

Chlorine (Cl2) gas Forms hypochlorous acid Dis infect drinking water,
(HClO), a strong oxidizing general disinfectant.
agent.

Silver Nitrate (Ag No3) Precipitates proteins. General antiseptic and

 used in the eyes of
newborns.

Mercuric chloride Inactivates proteins by Disinfectant although

reacting with sulfide occasionally used as an
groups. antiseptic on skin.

Detergents (eg: Quaternary Disrupts cell membranes. Skin antiseptics and

ammonium compounds) disinfectants.

Chemotherapeutic agents
Antimicrobial agents of synthetic origin useful in the treatment of microbial or viral
disease. Examples: sulfonilamides, isoniazid, ethambutol, AZT, chloramphenicol.
Antibiotics
Antimicrobial agents produced by micro organisms that kill or inhibit other micro
organisms. This is the microbiologist’s definition. A more broadened definition of an
antibiotic includes many chemical of natural origin which has the effect to kill pr inhibit
the growth of other types cells. Since most clinically useful anti biotics are produced by
micro organisms and are used to kill or inhibit infectious bacteria we follow classic
definition.
Antibiotics are low molecular weight (non- protein) molecules produced as
secondary metabolites, mainly by micro organisms that live in the soil. Most of these
micro organisms form some type of a spore or other dormant cell, and there is thought
to be some relationship between anti biotic production and the process of sporulation.
Among the molds, the notable antibiotic producers are penicillium and cephalosporium,
which are the main source of the beta lactam antibiotics. In the bacteria, the
actinomycetes, notable streptomyces species, produce a variety of types of anti biotics
including the aminoglycosides (eg: streptomycin), macrolides (eg: erythromycin) and
the tetracycline. Endospore forming bacillus species produce polypeptide anti biotics
such as polymyxin and bactracin.

Chemical class examples Biological source Spectrum Mode of action

(effective
against)

Beta – lactams Penicillin G, Penicillium notatum Gram positive Inhibits steps inc ell
Cephalothin and cephalosporium bacteria wall
(Penicillins and
sp. (peptidoglycan)
cephalosporins)
synthesis and
 murein assembly.

Aminoglycosides streptomycin Streptomyces griseus Gram positive Inhibit translation

and gram (protein synthesis)
negative
bacteria

glycopeptides vancomycin Streptomyces Gram positive Inhibits steps inn

orientales bacteria, esp. murein
staphylococcus (peptidoglycan)
aurues biosynthesis and
assembly

macrolides erythromycin Streptomyces Gram positive Inhibits

erythreus and gram translation(protein
negative synthesis)
bacteria not
enteric,.
Neisseria,
legionella,
mycoplasma

polypeptides polymyxin Bacillus polymyxa Gram negative Damages

bacteria cytoplasmic
membranes

Polyenes amphotericin Streptomyces Fungi Inactivate

nodosus membranes
containing sterols

tetracyclines tetracycline Streptomyces sp Gram positive Inhibit translation

and gram (protein synthesis)
negative
bacteria,
rickettsias

Chloramphenicol Chlorampheni Streptomyces Gram positive Inhibit translation

col venezuelae and gram (protein synthesis)
negative
bacteria
 METABOLISM IN BACTERIA
Microbial Metabolism
Metabolism refers the sum of biochemical reactions required for energy generation
and the use of energy to synthesize cellular materials.
The energy generation component is referred as catabolism and the build up of
macromolecules and cell organelles are referred as anabolism.
During catabolism, the energy is changed from one compound to
another and finally conserved as high energy bonds of ATP.
ATP is the universal currency for energy. When energy is required for anabolism, it
may be sent as high energy bonds of ATP which has the value of 8 kcal per mole.

Based on the source of carbon, the microbes can be divided into two groups namely,
autotrophs and heterotrophs. Autotrophs utilize CO2 as sole carbon source and
heterotrophs use organic carbon as sole carbon source.

I. Energy generation by heterotrophs

Heterotrophs use variety of carbon sources. Glucose is being the simple and wide
variety of microbes prefers it. The glucose can be taken up by bacterium through diffusion
and can be readily utilized. There are three possible pathways available in bacteria to use
glucose. All these path ways are fermentative type and substrate level phosphorylation
occurs.
• Embden-Meyerhof path way
• Phosphoketolase path way
• Entner – Doudoroff path way.

SUBSTRATE LEVEL PHOSPHORYLATION: (Fermentation)

The EMP pathway, phosphoketolase pathway and ED pathway end with one or two
ATP synthesis by substrate level phosphorylation. There won’t be any external source of
electron acceptor will come in these reactions.

A. Embden-Meyerhof path way

This is the path way of glycolysis most familiar and common to most of the
organisms. The path way is operated by yeast to produce alcohol and lactic acid bacteria to
produce lactic acid and several organic acids, gases, fatty acids, and alcohols. The path way
is as follows:
Glucose  2 pyruvate + 2 ATP + 2 NADH2
 After pyruvate is formed, if the organism is a respirative type, the pyruvate will go
to Krebs cycle and if the organism is fermentative, the reduction process ends with
organic acids, alcohols etc.

A model fermentation: After an intermediate product, a reduction takes place in

fermentation, whereas, if respiration, CO2 will be formed by complete oxidation through
Krebs’s cycle
(Note: After pyruvate, the reduction process leads to fermentation and complete oxidation
leads to respiration)
The Embden – Meyerhof path way can lead to a wide array of end products
depending on the path ways taken in the reductive steps after the pyruvate formation.
The following are some of the such fermentations:

Fermentation End products Model organism

Homolactic fermentation Lactic acid Lactobacillus
Mixed acid fermentation Lactate, acetate, Enterobater
formate, succinate
Butyric acid fermentation Butric acid, acetone Clostridium
acetobutylicum
Propionic acid fermentation Propionic acid Propionibacterium
Alcohol fermentation Ethanol Saccharomyces
B. Phosphoketolase path way (Heterolactic path way)
The phosphoketolase path way is distinguished by the key cleavage enzyme
phosphoketolase, which cleaves pentose to glyceroldehyde 3 phosphate and acetyl
phosphate. The path way ends with ethanol and lactic acid. Ex. Lactobacillus, Leuconostoc.
The overall reaction is,
Glucose  1 lactate + 1 ethanol + 1 CO2 + 1 ATP
This path way is useful in the dairy industry for preparation of kefir (fermented milk),
yogurt, etc.

C. Entner – Doudoroff pathway

Only few bacteria like, Zymomonas mobilis employ the ED pathway. The path way is
as follows:
The overall reaction is
Glucose  2 ethanol + 2 CO2 + 1 ATP
The alcohol productivity of Zymomonas is higher than yeast because of this fermentative
pathway.
( Note : All the three pathways are end with 1 or 2 ATP by substrate level phosphorylation
by means fermentation)

OXIDATIVE PHOSPHORYLATION (Respiration)

If the organism is a respiratory type (that means complete oxidation of glucose), it
needs four essential metabolic components for their respiration and oxidative
phosphorylation.
a. Tricarboxylic acid cycle (also known as citric acid cycle or Kreb’s cycle) The
pyruvate formed during glycolysis will be completely oxidized to 3 CO2 by the use of this
cycle. During oxidation of one pyruvate through TCA cycle, 4 NADH2, 1 FADH2 and 1 GTP
are produced along with 3 CO2.
b. A membrane and associated Electron Transport System (ETC) The electron
transport chain is a sequence of electron carriers transport the electrons to a terminal
electron acceptor. During this flow of electron in the membrane, a proton motive force
across the membrane leads to formation ATP (is referred as electron transport
phosphorylation).
c. An outside electron carrier: for aerobic respiration, O2 is the terminal electron acceptor
and reduced to H2O. This is normal for higher organisms. But in anaerobic bacteria, the
terminal electron acceptor may be of nitrite, nitrate, sulphate or carbon dioxide.
d. A membrane bound ATPase enzyme: The proton motive force developed during ETC
leads to formation of ATP by enzyme ATPase present in the membrane. (As in the diagram)
The table shows some aerobic and anaerobic respirations with specific examples:

Terminal End product Process name Organism

electron
acceptor
O2 H 2O Aerobic respiration Streptomyces
NO3 NO2, N2 Denitrification Pseudomonas denitrificans
SO4 S or H2S Sulphate reduction Desulfovibrio desulfuricans
Fumarate Succinate Anaerobic respiration Escherichia
CO2 Methane (CH4) Methanogenesis Methanococcus

In aerobic organisms, the terminal electron acceptor will be of O2. In some

anaerobic organisms, after the electron transport chain, instead of O2, some inorganic
compounds like sulphate, nitrate or some organic compounds like fumarate act as terminal
electron acceptor. Such type of respiration is referred as anaerobic respiration and the
normal O2 mediated respiration is referred as aerobic respiration. The above table shows
some anaerobic respiration with some terminal electron acceptors. The process is named
based on the compounds as sulphur reduction, denitrification and methanogenesis.
Energy generation by autotrophs
Autotrophs use CO2 as their sole carbon source. There are two types such as
photoautotrophs and chemoautotrophs. Photoautotrophs use light as energy source and CO2
as carbon source. Chemoautotrops use chemicals (especially inorganic) as energy source
and CO2 as carbon source.
I. Energy and carbon assimilation by photoautotrphs: (Photoautotrophy)
Phototrophs use sunlight to produce ATP through phosphorylation, referred as
photophosphorylation. The phototrophs convert the light energy to chemical energy (ATP)
through the process called photosynthesis.
Photosynthesis is a type of metabolism in which catabolism and anabolism occur as
sequence. The catabolic reaction (energy generating process) of photosynthesis is light
reaction in which the light energy is converted to chemical energy (ATP) and electrons or
reducing powers (NADPH). The anabolic reaction (macromolecule synthesis) of
photosynthesis is dark reaction in which CO2 is converted to organic molecules
(carbohydrates), which is also called as CO2 fixation.
 For conversion of light energy to ATP, the bacteria possess light harvesting pigments.
They are chlorophyll a, carotenoids, phycobiliproteins (which are present in cyanobacteria)
and bacteriochlorophyll (which are present in purple sulphur bacteria). In bacteria, there are
two types of light reactions (conversion of light to ATP) and two types of CO2 fixation occur.

A. Light reaction (Photophosphorylation)

For photophosphorylation, light harvesting pigments, a membrane electron transport
chain, source of electron (electron donor) and ATPase enzymes are required. Two types of
photophosphorylations occur during photosynthesis. They are cyclic photophosphorylation
and non-cyclic photophosphorylation.
 In plant and cyanobacteria, both cyclic and non-cyclic photophosphorylation occurs
whereas in purple bacteria, the cyclic photophosphorylation only occurs.
 In plant and cyanobacteria, the electron source is water, by photolysis, H2O split into
+
H and O2 and during the process, O2 is evolved and referred as oxygenic photosynthesis
 Since, the sulphur bacteria is an anaerobic bacterium, they use H2S instead of H2O as
electron donor. Since, there won’t be any O2 evolution during photosynthesis, referred as
anoxygenic photosynthesis.
Difference between plant and bacterial photosynthesis
organisms Plant photosynthesis Bacterial photosynthesis
plants, algae, purple and green bacteria
cyanobacteria
type of chlorophyll chlorophyll a bacteriochlorophyll
absorbs 650-750nm absorbs 800-1000nm
Photosystem I present present
(cyclic photophosphorylation)
Photosystem II (noncyclic present absent
photophosphorylation)
Produces O2 Yes (Oxygenic) No (Anoxygenic)
Photosynthetic electron donor H 2O H2S, other sulfur compounds
or certain organic compounds

1. The oxygenic photophosphorylation

The end product of the light reaction is ATP, NADPH and O2. The ATP and NADPH,
the energy and electron sources thus produced were used for dark reaction.
2. The anoxygenic photo phosphorylation
 The anoxygenic photo phosphorylation will take palce as in the image and the end
product of the light reaction is ATP, NADPH and Sulphur. The ATP and NADPH, the energy
and electron sources thus produced were used for dark reaction.

B. Dark reaction (CO2 fixation)

The dark reaction in which the ATP and NADPH were used as energy and electron
sources to fix the CO2 as carbohydrates. The pathway involved in the dark reaction is
Calvin cycle, by which the CO2 is fixed as phosphoglyceic acid and lead to formation of
many sugars. The enzyme RuBiSCO is the key enzyme for this process.
The following pathway shows the Calvin cycle and the formation of key monomers for
anabolic reactions such as hexose phosphate – polysaccharides; pyruvic acid – amino
acid and fatty acid; pentose phosphate – DNA and RNA.
 A complete model of light and dark reaction of photosynthesis

Another way of CO2 fixation by phototrophs

In phototrophs, the electron and energy were derived form sunlight and carbon from
CO2 fixation through Calvin cycle. But some bacteria may derive electron and energy from
sunlight and fixes CO2 by some other path way, not the Calvin cycle. The example is
Photosynthetic green bacteria (Chlorobium). They derive NADPH and ATP through cyclic
phosphorylation, but CO2 fixation is by reverse TCA cycle. Since TCA cycle is amphibolic
pathway (referring the cycle can operate in both the directions), it can also be used to fix
the carbon-di-oxide if operated reversely. The pathway is as follows:

Another way of CO2 fixation is by methanogens: They use CO2 as terminal electron
acceptor and forms CH4 (methane). They also fix by acetyl CoA pathway for fixing CO2.
Synopsis:
Organism Light reaction/ATP generation Dark reaction/CO2
fixation
Cyanobacteria Cyclic and non-cyclic Calvin cycle
(Nostoc), plant photophosphorylation
and alga Oxygenic photosynthesis
Purple bacteria Cyclic and non-cyclic Calvin cycle
(Chromatium) photophosphorylation
Oxygenic photosynthesis
Green bacteria Cyclic and non-cyclic Reverse TCA cycle
(Chlorobium) photophosphorylation
Oxygenic photosynthesis

II. Energy and carbon assimilation by Chemoautotrophs: (Chemoautotrophy)

Since the chemoautotrophs use inorganic chemicals for their energy and electron
source, they are referred as chemolithotrophs or chemolithotrophic autotrophs.
These organisms remove electron from an inorganic substance and put them through
electron transport chain for ATP synthesis (through electron transport phosphorylation). At
the same time, the electrons were also flow through reverse electron transport chain
and with the end product of NADPH. These ATP and NADPH were used for CO2 fixation
through Calvin cycle. These bacteria are obligate aerobic organisms. Some examples of
the chemolithotrophs are as follows:
Groups of chemolithotrophs

Physiological group Energy Oxidized Organism

source end product
hydrogen bacteria H2 H 2O Alcaligenes, Pseudomonas
nitrifying bacteria NH3 NO2 Nitrosomonas
nitrifying bacteria NO2 NO3 Nitrobacter
sulfur oxidizing bacteria H2S or S SO4 Thiobacillus, Sulfolobus
2+ 3+
iron oxidizing bacteria Fe Fe Gallionella, Thiobacillus
The following diagram shows energy generation and CO2 fixation by different
chemolithotrophs:

 
 ATP GENERATION

The energy captured within ATP can then be harnessed to create order in the form of
biosynthetic reactions. In a hypothetical enzyme reaction that converts substrates A−H and
B−OH to A−B and H2 O, the energy from ATP hydrolysis is first used to convert B−OH to a
higher-energy intermediate, B−O−PO4. This compound is only transiently formed, with the
energy released during its decay used by the enzyme to form A−B. Thus, the energy
released from the ATP hydrolysis reaction (large −_G) is coupled to the synthesis reaction
(large +_G). In this way, the cell can progressively create order.

Electron transport system (ETS)

• Although cells could transfer electrons directly from NADH to oxygen, this would liberate
all energy in NADH directly as heat.

• NADH possesses lots of energy. If electrons are transferred directly to oxygen:

• NADH + O2 NAD + H2O, delta Go' = - 218 kjoules/mole
• If NADH has ~218 kjoules of energy, and it only takes 30.5 kjoules to make one ATP,
could conceivably make 218/30.5 = ~ 7 ATP per NADH if energy conversion were
100% efficient.

• In practice, cells have evolved ways to get up to 40% efficiency (~ 3 ATP/NADH) under
optimal circumstances.

• Electron transport system (ETS) = membrane-bound pathway transferring electrons from

organic molecules to oxygen.

• ETS moves both electrons and protons:electrons are passed from carrier to carrier in the
membrane, while protons are moved from inside to outside of membrane

• Net result: electrons enter ETS from carriers like NADH or FADH, wind up at terminal
oxidase, get attached to oxygen.

• ETS consists of 4 complexes, connected by mobile carriers (Coenzyme Q, cytochrome c)

that shuttle between complexes in membrane
Specific carriers of ETS:
• mitochondria (in eukaryotes): NADH ---> (Flavoprotein Iron sulfur proteins Quinone
cytochrome b cytochrome c cytochrome a cytochrome a3 oxygen
• bacteria (prokaryotes) have different ETS carriers, shorter chains. In E. coli, can have two
different terminal oxidases, one functions at high oxygen levels, one at lower oxygen
levels. Cytochromes involved include: b558, b595, b562, d, and o
• proton gradient and oxidative phosphorylation (oxphos)

Chemiosmotic hypothesis (Peter Mitchell, 1961)

• As electrons flow through ETS, at certain steps protons (H+) are moved from inside to
outside of the membrane.

• This builds up proton gradient; since + charges are removed from inside of cell, - charge
remains inside, mainly as OH- ions.

• pH just outside membrane can reach 5.5, pH just inside membrane can reach 8.5 --->
difference of 3 pH units, or 1000x concentration differential of H+ across membrane.
 This represents potential energy stored up in proton gradient = proton motive force.

• Membrane is basically impermeable to protons, so gradient doesn't get squandered away

by leaky reentry.

• ATP synthase protein complex contains only channels for proton entry. As protons push in
through channel, the base rotates. Specific binding sites allow ADP + Pi ATP. This
can be called chemiosmotic phosphorylation (assuming chemiosmotic hypothesis is
correct), or oxidative phosphorylation (makes no assumption about mechanism.

Oxidative phosphorylation
Differences between respiration in mitochondria (eukaryotes) and bacteria
(procaryotes)

1. In Eukaryotes:

• ETS located in inner mitochondrial membrane. Proton gradient develops

across inner mitochondrial membrane.

• Mitochondria are very efficient at generating proton gradient. Can measure

how many ~P bonds (in ATP) are made for each O2 consumed = P/O ratio.

• With NADH as electron donor, P/O ratio can be 3 (means 3 ATP made per
NADH).

• But with FADH as electron donor, P/O ration only 2 (fewer protons are
transported, less proton gradient).

• Overall efficiency of respiration in mitochondria: ~ 40% (means that about

40% of energy in glucose actually gets converted to ATP).

2. In Prokaryotes:

• ETS located in cytoplasmic membrane. Proton gradient develops across

this membrane.

• Bacteria are not as efficient. ETS chains are shorter, P/O ratios are lower.

• As a ballpark estimate, P/O ratios for NADH are only ~2. Overall efficiency
of glucose oxidation is closer to 28%, not 40%.
Inhibitors of Oxidative Phosphorylation

• Several chemicals can block electron transfer in ETS, or transfer of electrons to

oxygen. All are strong poisons. Some examples:

• Carbon monoxide -- combines directly with terminal cytochrome oxidase,

blocks oxygen attachment

• Cyanide (CN-) and Azide (N3-) bind to cytochrome iron atoms, prevent
 electron transfer.

• Antimycin A (an antibiotic) inhibits electron transfer between cyt b and c.

Anaerobic respiration

• Use of acceptors other than oxygen.

• Most common in bacteria. Most alternative electron acceptors are

inorganic molecules, but some organic molecules can serve.

• As with aerobic respiration, anaerobic respiration uses ETS, membrane

localization, proton gradient, and ATP synthase.

• Processes are of great importance both ecologically and industrially.

Anaerobic respiration
Nitrate (NO3-)

• Process called denitrification. Also called dissimilative nitrate reduction.

Reduced waste products are excreted in significant amounts.

• Redox potential is + 0.42 v (compared to + 0.82 v for oxygen). So

organisms respiring anaerobically gain less energy than with oxygen.

• Requires new terminal oxidase called nitrate reductase. Enzyme is

repressed by oxygen, synthesis turned on in absence of oxygen.

• Process can have several steps, proceed in two different directions:

(A) nitrate (NO3-) nitrite (NO2-) ammonia (NH3)
(B) nitrate (NO3-) nitrite (NO2-) nitrous oxide (N2O) dinitrogen gas (N2)

• Second process is major pathway for loss of nitrogen compounds from

soil, return of nitrogen to atmosphere.

• Pseudomonas species are common denitrifiers, widespread in soils. When

fertilized soils become flooded, oxygen is rapidly depleted, pseudomonads
switch to anaerobic respiration and can use up soil nitrate, leaving field in
unfertile state.

• Note: Studied this in lab. Media must contain nitrate in addition to

nutrients, otherwise won't work. Also, in scavenger hunt at end of course,
one target microbe will be Pseudomonas, enrichment culture depends on
its ability to grown anaerobically using nitrate reduction.
Sulfate (SO42-)

• Process called sulfate reduction.

 • Sulfate (SO42-) Hydrogen Sulfide (H2S)

• Small group of bacteria carry out this reaction; all obligate anaerobes.

• Have unique cytochrome c3.

• Sulfate is common in sea water. Often, H2S combines with iron, forms
insoluble FeS black sediments. Common in estuaries.
Carbon dioxide (CO2)

• One of most common inorganic ions.

• Methanogens: most important group of CO2 reducers. Obligate

anaerobes, archaebacteria. Produce methane as waste product.

• Reaction: CO2 + H2 + H+ CH4 + H2O

• Note: reaction also requires Hydrogen gas. Methanogens typically live

alongside bacteria that produce hydrogen by fermentation, remove
hydrogen as it is made.

TCA cycle: further catabolism of pyruvate

Formation of acetyl-CoA

• Oxidation of pyruvate (3-C) + NAD+ Acetyl-CoA (2-C) + CO2 + NADH

• Carried out by pyruvate dehydrogenase (multi-enzyme system)

• Note: Acetyl-CoA can also be produced by breakdown of lipids or certain

amino acids -- important focal point of central metabolism

• net effects of TCA cycle

• To start cycle:
• Acetyl-CoA (2-C) + oxaloacetate (4-C) citric acid (6-C)

• Subsequent steps:
• Convert citrate to isocitrate (still 6-C)

• Oxidize alpha-ketoglutarate (5-C) + CO2 + NADH

• Oxidize succinyl-CoA (4-C) + CO2 + NADH

• SLP reaction: succinyl-CoA (4-C) + GDP succinate (4-C) + GTP (Note:

GTP can be interconverted with ADP to form ATP)

• Oxidize fumarate (4-C) + FADH2 -- convert fumarate to malate

• (6)oxidize again oxaloacetate (4-C) + NADH

• Net yield: Acetyl-CoA (2-C) + 3 NAD+ + FAD 2 CO2 + 3 NADH + FADH2

 + ATP

• TCA cycle completes the oxidation of carbons in pyruvate to most oxidized

form (CO2); removes electrons originally in C-H bonds to electron carriers
NADH and FADH for use in respiration machinery.
Catabolism of substances other than glucose

• Many other possible C-sources for catabolism beside glucose. In general, must convert
these into molecules that can enter into central metabolism, either in glycolysis or
TCA cycle.

1. carbohydrates

▪ Most abundant C-sources in most environments, most in various

polysaccharides (cellulose, starch, lignin, etc.)

▪ To gain access to sugars, must first secrete hydrolytic enzymes that break
down glycosidic bonds in polysaccharides, produce mono- and
disaccharides that can be transported into cells.

▪ Starch, glycogen -- easily hydrolyzed by amylases

▪ Cellulose -- difficult to digest, very insoluble, tightly folded. Many fungi,

some bacteria produce cellulases.

▪ Agar -- some marine bacteria produce agars

▪ Once mono- or disaccharides are available, they are transported into cell,
converted into some typical glycolytic intermediate such as glucose-6-
phosphate, catabolized by glycolytic enzymes.

2. lipids

▪ Biological lipids common as triglycerides, diglycerides.

▪ To catabolize, bacteria secrete lipases, hydrolyze glycerides to free fatty

acids and glycerol.

▪ Fatty acids attacked by Beta-oxidation pathway.

▪ Using FAD and NAD+ to remove electrons, 2-C units are removed as
Acetyl-CoA, feed directly into central metabolism at TCA cycle entry.
Glycolysis pathway not involved (except for use in synthesizing sugars
needed for cell wall, running sections of pathway in reverse).

3. proteins

▪ Proteins must first be hydrolyzed by protease enzymes, to get individual

amino acids which can be transported into cells.

▪ Amino acids all have common structure: NH2 - RCH - COOH.

▪ 1st step in catabolism is to remove amino group (deamination), often by
swapping it with another substrate (transamination).

▪ Typical example: glutamic acid (an AA) + pyruvate alpha-ketoglutarate +

alanine (= pyruvate + amino group). Now alpha-KG can be oxidized in
TCA cycle, since it is a TCA cycle compound.

▪ As excess amino groups accumulate, must be secreted as waste products,

possibly as ammonium ion (leads to alkaline pH).
 MICROBIAL METABOLISM: AUTOTROPHS

Overview of Autotrophy
• Imagine being hungry, walking outside, taking off your shirt, lying in the sun for a few hours,
becoming totally full (fat even!), and being done eating. No stores, no lines, no choices,
just sunlight --- and the machinery of an autotroph --- and some CO2 and a couple of
other requirements (water --- and H2S or Hydrogen gas, if you happen to be an anerobe)
• Autotroph = gets all carbon from CO2, organic C not required (for C-source). Use special
metabolic cycle: Calvin-Benson cycle
• Refers to C-source only; some organisms still require organic C as energy source

Calvin-Benson cycle
• Each CO2 is added to a 5-C acceptor molecule (ribulose 1,5 bis-phosphate)
• Immediately split into two 3-C molecules (3-phosphoglyceric acid)
• Must add phosphate group (from ATP) and hydrogen (from NADPH) to get reduced product,
3 - phospho-glyceraldehyde (PGA)
• Cannot take all (PGA) as product --- must regenerate some more acceptor to keep cycle
going. How?
• Take 5 PGA molecules (5 x 3C = 15 C atoms). Rearrange through series of reactions to make
3 5 - C molecules (still 15 C atoms). Add ATP to each, make 3 acceptor molecules
(ribulose 1,5 bis-phosphate)
• Net result: To get 1 PGA (3-C) as reduced product, need 3 CO2 molecules, added to 3
acceptor molecules ----> six 3 - C molecules, use 6 ATP and 6 NADPH ----> 6 PGA
molecules; five of these are used to regenerate acceptor molecules (+ 3ATP), one PGA
can leave cycle and be used by cell.

Summary
• Actual cyce exports 3-C reduced molecules: look at balanced equation:
3 CO2 + 9 ATP + 6 NADPH -----> 3-phospho-glyceraldehyde (PGA) + 9 ADP + 9
NADP+
• Often want to look at balanced equation relative to 6C synthesis. Must multiply all terms in
balanced equation above by two (since 2 PGA ~ 1 glucose)
6 CO2 + 18 ATP + 12 NADPH -----> glucose + 18 ADP + 12 NADP+
• Note for reaction: glucose + O2 ----> 6CO2 + 6 H2O; delta Go'= - 688kcal/mole
• If each ATP contains ~7.3 kcal/mole (from delta Go' for hydrolysis) and each NADPH contains
~54 kcal/mole (from delta Go' for oxidation), then to make glucose costs 780 kcal/mole,
more than the energy available by oxidizing glucose.
• Conclusion: making sugar is expensive! Cell needs to supply large quantities of ATP and
NADPH.

Chemolithotrophs
Hydrogen Bacteria
• Gain energy by oxidizing hydrogen gas:
• H2 + NAD+ -------(hydrogenase enzyme)------> NADH + H+
• alternative: electrons can be donated directly to ETS chain, bypassing NAD
• Note: only need one special enzyme to carry this step out: hydrogenase.
• Many different genera of bacteria include members that can induce hydrogenase. When
hydrogen disappears, back to heterotrophic life. Hydrogen bacteria are usually
facultative chemolithotrophs.

Sulfur Bacteria
• Called "colorless" in contrast to chlorophyll-containing sulfur bacteria, usually green or purple
• Oxidize sulfur compounds: Example: Thiobacillus thiooxidansthiosulfate: S2O3= -----> SO4=free
sulfur: 2 So + 2 H2O + 3 O2 -----> 2 H2SO4
• Note product: sulfuric acid!! Cells can grow even in pH 0 (1M sulfuric acid). But cell internal
pH is ~7, so difference across membrane can be 6 or 7 pH units.
• Acid mine drainage: common in Western Penn., E. Ohio, W. Virginia. Rivers can run rust red.
Mines have been major sources of pollution. Water seeps in, sulfur deposits exposed
during coal mining allow microbial growth ------> megatons of H2SO4
• Sulfuric acid leaches out, dissolves iron, precipitates in river with bicarbonate to form rusty
deposits.
• Quantities involved: Ohio River carries 100 million tons of 98% conc. H2SO4 per year.
• To cure problem, must seal up old mines, prevent oxygen access. Also strip mines must be
promptly covered up once mining is done to block access of microbes and oxygen to
sulfur.
• Value of this reaction:
(a) farmers or gardeners can dump free S on alkaline soil, bacteria will produce acid
(b) miners can use process to recover Cu from low grade ores, where smelting is not
 economical. Pile up mine "tailings" with copper ore; scrap shallow hole and fill with
water. If tailings contain S, microbes will produce H2SO4. Now pump the acid over the
tailings, Cu will be leached out and accumulate as soluble ions in acid pool. Eventually
process the acid, recover Cu.

Nitrifying Bacteria
• Very important soil organisms -- process all ammonia, nitrite in soils, break down amino acids,
nitrogen bases ---> ammonia (NH3)
• Two different groups: one oxidizes ammonia, one oxidizes nitrite
• Ex. 1: Nitrosomonas: 2 NH3 (ammonia) + 3 O2 -----> 2 HNO2 (nitrite) + 2 H2O
• Ex. 2: Nitrobacter: 2 HNO2 (nitrite) + 2 O2 -----> 2 HNO3 (nitrate)
• Note potential problem: redox potential for nitrite as electron donor is + 0.42 v., so can easily
pass electrons down to oxygen at + 0.82 v., reaction will be spontaneous. Electrons can
be passed through an electron transport system, make ATP by chemiosmotic
phosphorylation.
• BUT --- how to make NADPH? (Remember, this an autotroph, needs both ATP and NADPH
to grow). How to get NADPH? The redox potential is much higher than nitrite.
• Solution: Reverse electron transport. Accumulate enough proton gradient by oxidation of
nitrite to force electrons back to carriers with higher redox potentials, all the way back to
NADH ---> NADPH. This works as long as concentrations of reduced forms are kept very
low, and NADPH is used up immediately to make glyceraldehyde-3-phosphate. See
handout
• This is very inefficient process. Nitrobacter can have 18 hour generation time. But it has no
competition, so what's a little extra time?

Iron Bacteria
• Curious discovery: Ferrobacillus ferrooxidans. Carries out oxidation of iron: Fe++ (ferrous) ---->
Fe+++ (ferric) + e-
• Originally thought bacteria get energy from oxidation, make ATP. But redox potential of Fe
oxidation is + 0.78 v., and redox potential for oxygen is + 0.86 v., so delta Eo' for aerobic
respiration is only -0.08 v., calculated delta Go' is much less than the 7.3 kcal/mole
needed to make ATP. How does this organism grow?
• It only grows in very acidic habitats, pH less than 3. Found with Thiobacillus thiooxidans,
bacterium that produces sulfuric acid. Ferrobacillus lives off the pH gradient created by
 acidic pH. This maintains very high proton gradient. As H+ flows in, ATP gets made. But
need to get rid of H+ inside, keep internal pH at 7. Use Fe++ as electron donor to oxygen,
combine with H+ to form water, get rid of outside cell. Iron functions as electron supplier
to get rid of protons.
• Cells process an enormous amount of iron for very small yields of energy. Fe+++ reacts with
OH- ions to form insoluble precipitate, Fe(OH)3, reddish yellow color.

Phototrophs
• Use energy from sunlight to get high energy electrons (attached to carriers high on redox
tower). Use CO2 and Calvin-Benson cycle to make all organic molecules.
• Critical molecules: photon absorbers = bacteriochlorophylls. Several different varieties. Light
is trapped by a patch of pigments = "antenna field", gets passed around to a "reaction
center" where an electron is released from Mg++ ion with high energy, passed to electron
transport system -- from this point, can use electron transport systems to generate
proton gradients, make ATP.
• Problem: need to make not only ATP (available from proton gradient), but also NADPH. How
to obtain?
• Two solutions:
use a reduced molecule with high redox potential like hydrogen gas (H2) or hydrogen
sulfide (H2S) to pass electrons to NADP+. Light not needed for this.
use a reduced molecule with low redox potential like water to release electrons and H+
ions. Need lots of energy to drive this reaction, so need an extra step. Light is
needed for this.

Anaerobic photosynthetic bacteria

• Three common groups:
Purple bacteria Exs: Chromatium vinosum, Thiospirillum jenense
Purple nonsulfur bacteria. Exs: Rhodospirillum rubrum, Rhodobacter sphaeroides
vannielii
Green sulfur bacteria (many are actually brown) Exs: Chlorobium limicola,
Prosthecochloris aestuarii,
• Notes: in both groups, electrons released by light travel through electron transport systems
back to the original photosystem = cyclic electron flow. Proton gradient is produced, ATP
is made as protons flow back through ATP synthase molecules. Specific carriers are
 different.
• To make NADPH, need reduced electron donor.(1) in purple bacteria, can use organic
molecules (e.g. fumarate), or H2 for non-sulfur bacteria; or can use H2S or H2 for purple
sulfur bacteria. Sulfur accumulates inside cells when H2S is used, hence the name.(2) in
green sulfur bacteria, can use H2S, or H2. Sulfur accumulates outside cells.

Aerobic photosynthetic bacteria = cyanobacteria

• includes both prokaryotes (cyanobacteria, formerly called blue-green bacteria) and
eukaryotes (algae, green plants)
• Two photosystems are needed, not one as in anoxygenic photosynthesis. Why?
• Source of NADPH = electrons removed from photosystem I ---> excited by light to high
redox potential, passed to ferredoxin, then directly to NADP+ -----> NADPH
• Now photosystem I has + charge, can't supply any more electrons. Can't have this, so
replace electrons from another photosystem II (see handout diagram), also energized by
light. During this process, electrons flow through ETS system and make a proton
gradient ( ------> ATP by chemiosmotic phosphorylation). But electrons aren't flowing
back to same place they started from --- this is non-cyclic electron flow. Path resembles
a letter "Z", so often called "Z-scheme" photosynthesis.

FERMENTATION
• Fermentation -- oxidation of an organic compound in the absence of external electron
acceptor (no oxygen required). Uses SLP (substrate-level phosphorylation).
• Respiration -- oxidation of an organic compound where oxygen is the final electron
acceptor. Uses ETS (electron transport system) as well as SLP.
• Anaerobic respiration (unique to bacteria) -- oxidation of organic compounds where an
external substrate other than oxygen serves as final electron acceptor. Exs: nitrate,
sulfate, carbon dioxide.
Lactic acid fermentation
• pyruvate + NADH lactic acid + NAD+
• found in many bacteria: lactic acid bacteria, Bacillus, also in some protozoa, water
molds, even human skeletal muscle
• Responsible for souring of milk products yogurt, cheese, buttermilk, sour cream, etc.
Excellent keeping properties.
• Some bacteria produce only lactic acid = Homolactic fermenters
 • Other bacteria produce other products as well; ethanol, CO2, lactate, etc. = Heterolactic
fermenters

Alcoholic fermentation
• pyruvate acetaldehyde + CO2
• acetaldehyde + NADH ethanol + NAD+
• Found in many fungi, yeasts, some bacteria.
• Very important in human applications. Bread, alcoholic spirits
Formic acid and mixed acid fermentations
• pyruvate (3-C) + CoA Acetyl-CoA (2-C) + formic acid (1-C)
• HCOOH CO2 + H2
• found in many bacteria, very common in enterics (Gram-negative facultative anaerobic rods,
include E. coli and other common intestinal tract denizens).
useful in identification: 2 common variants
Mixed acid fermentation
Some bacteria use several pathways, produce ethanol, formic acid, acetic acid, lactic acid,
succinic acid, CO2, and H2. Note lots of acid, lower pH than many other fermentations. Note:
ATP yield via mixed acid is ~2.5 ATP/glucose, a bit higher than straight lactic acid fermentation
Butanediol fermentation
Butanediol produced, also much more CO2, and H2

Roles of fermentation in nature

• Fermentations play major role.
• large part of cellulose ingested by herbivores is excreted in undigested form.
• Wherever organic matter accumulates, bacteria can grow and remove oxygen (by
respiration), leading to anaerobic conditions that favor fermentation.
• Even in lab cultures (test tubes of media), bacteria eat up all available oxygen, rely
largely on fermentation unless vigorous aeration is maintained! Bacteria are pigs, gorge
themselves at every opportunity!
• Beside bacteria, fermentations also carried out be protozoa, fungi, even animal muscle
tissues (only works as temporary energy supplement).

What substances can be fermented?

• must have intermediate oxidation state (o.s.)
 • if totally oxidized (-CO)n cannot be fermented
• if totally reduced (-CH2)n, cannot be fermented
• must be convertible to a substrate for substrate level phosphorylation (usually into some
glycolytic step)
• Many sugars can be fermented. Also amino acids (e.g. by Clostridia, oxidizing one
amino acid and using a different amino acid as electron acceptor.)

Respiration
• Use an external electron acceptor. Oxygen as prototype.
• The "problem" with fermentation is that, by using an organic molecule as a terminal
electron acceptor to be discarded as waste, cell is losing out on potential to further
oxidize organic molecule, get more energy.
• Alternative solution is to use some non-organic molecule that has a low redox potential,
can accept electrons and become some reduced molecule. Oxygen is perfect for this,
has extremely low redox potential, and becomes reduced to water, the "perfect" waste
product for an aqueous environment.
• To transfer electrons (and protons, H+) to oxygen, need special oxidase enzyme. In
mitochondria, this is a cytochrome, cyt a. In bacteria, different cytochromes; in E. coli,
cyt o or d.
 BACTERIOPHAGES: STRUCTURE AND PROPERTIES OF BACTERIAL VIRUSES

Bacteriophage (phage) are obligate intracellular parasites that multiply inside bacteria by
making use of some or all of the host biosynthetic machinery (pathmicro.med.sc.edu/ppt). The
term is commonly used in its shortened form, phage. Interestingly, Bacteriophages are much
smaller than the bacteria they destroy.Phages are estimated to be the most widely distributed
and diverse entities in the biosphere. Phages are ubiquitous and can be found in all reservoirs
populated by bacterial hosts, such as soil or the intestines of animals. One of the densest
natural sources for phages and other viruses is sea water. They have been used for over 60
years as an alternative to antibiotics, however, this much controversial area of research
(http://www.phages.org).
Typical phages have hollow heads (where the
phage DNA or RNA is stored) and tunnel tails, the tips of
which have the ability to bind to specific molecules on
the surface of their target bacteria. The viral DNA is then
injected through the tail into the host cell, where it directs
the production of progeny phages often over a hundred
in half an hour. These "young" phages burst from the
host cell (killing it) and infect more bacteria.

Composition of bacteriophages
Although different bacteriophages may contain different materials they all contain nucleic
acid and protein. Depending upon the phage, the nucleic acid can be either DNA or RNA but not
both and it can exist in various forms. Bacteriophages have been classified as:
 The nucleic acids of phages often contain unusual or modified bases. These modified
bases protect phage nucleic acid from nucleases that break down host nucleic acids during
phage infection. The size of the nucleic acid varies depending upon the phage. The simplest
phages only have enough nucleic acid to code for 3-5 average size gene products while the
more complex phages may code for over 100 gene products.
The number of different kinds of protein and the amount of each kind of protein in the
phage particle will vary depending upon the phage. The simplest phages have many copies of
only one or two different proteins while more complex phages may have many different kinds.
The proteins function in infection and to protect the nucleic acid from nucleases in the
environment. Phages are also commonly employed in gene cloning, especially those exhibiting
lytic and lysogenic cycles (http://www.web-books.com)
Structure of bacteriophages
Bacteriophage comes in many different sizes and shapes. The basic structural features of
bacteriophages are (which depicts the phage called T4)
1. Size - T4 is among the largest phages; it is approximately 200 nm long and 80-100 nm wide.
Other phages are smaller. Most phages range in size from 24-200 nm in length.
2. Head or Capsid - All phages contain a head structure which can vary in size and shape.
Some are icosahedral (20 sides) others are filamentous. The head or capsid is composed of
many copies of one or more different proteins. Inside the head is found the nucleic acid. The
head acts as the protective covering for the nucleic acid.
3. Tail - Many but not all phages have tails attached to the phage head. The tail is a hollow tube
through which the nucleic acid passes during infection. The size of the tail can vary and some
phages do not even have a tail structure. In the more complex phages like T4 the tail is
surrounded by a contractile sheath which contracts during infection of the bacterium. At the end
of the tail the more complex phages like T4 have a base plate and one or more tail fibers
attached to it. The base plate and tail fibers are involved in the binding of the phage to the
bacterial cell. Not all phages have base plates and tail fibers. In these instances other structures
are involved in binding of the phage particle to the bacterium.
 Infection of Host Cells (http:// biology.about.com/od/virology)

A. Adsorption
The first step in the infection process is the adsorption of the phage to the bacterial cell.
This step is mediated by the tail fibers or by some analogous structure on those phages that
lack tail fibers and it is reversible. The tail fibers attach to specific receptors on the bacterial cell
and the host specificity of the phage (i.e. the bacteria that it is able to infect) is usually
determined by the type of tail fibers that a phage has. The nature of the bacterial receptor varies
for different bacteria. Examples include proteins on the outer surface of the bacterium, LPS, pili,
and lipoprotein. These receptors are on the bacteria for other purposes and phages have
evolved to use these receptors for infection.
B. Irreversible attachment
The attachment of the phage to the bacterium via the tail fibers is a weak one and is
reversible. Irreversible binding of phage to a bacterium is mediated by one or more of the
components of the base plate. Phages lacking base plates have other ways of becoming tightly
bound to the bacterial cell.VIE
C. Sheath Contraction
The irreversible binding of the phage to the bacterium results in the contraction of the
sheath (for those phages which have a sheath) and the hollow tail fiber is pushed through the
bacterial envelope. Phages that don't have contractile sheaths use other mechanisms to get the
phage particle through the bacterial envelope. Some phages have enzymes that digest various
components of the bacterial envelope.
D. Nucleic Acid Injection
When the phage has gotten through the bacterial envelope the nucleic acid from the
head passes through the hollow tail and enters the bacterial cell. Usually, the only phage
component that actually enters the cell is the nucleic acid. The remainder of the phage remains
on the outside of the bacterium. There are some exceptions to this rule. This is different from
animal cell viruses in which most of the virus particle usually gets into the cell. This difference is
probably due to the inability of bacteria to engulf materials.
 LYTIC AND LYSOGENIC CYCLES - PHAGE MULTIPLICATION CYCLE

A. Definition - Lytic or virulent phages are phages which can only multiply on bacteria and kill
the cell by lysis at the end of the life cycle.

Lytic or Virulent Phages

a. Eclipse period - During the eclipse phase, no infectious phage particles can be found either
inside or outside the bacterial cell. The phage nucleic acid takes over the host biosynthetic
machinery and phage specified m-RNA's and proteins are made. There is an orderly expression
of phage directed macromolecular synthesis, just as one sees in animal virus infections. Early
m-RNA's code for early proteins which are needed for phage DNA synthesis and for shutting off
host DNA, RNA and protein biosynthesis. In some cases the early proteins actually degrade the
host chromosome. After phage DNA is made late m-RNA's and late proteins are made. The late
proteins are the structural proteins that comprise the phage as well as the proteins needed for
lysis of the bacterial cell.
b. Intracellular Accumulation Phase - In this phase the nucleic acid and structural proteins
that have been made are assembled and infectious phage particles accumulate within the cell.
c. Lysis and Release Phase - After a while the bacteria begin to lyse due to the accumulation
of the phage lysis protein and intracellular phage are released into the medium. The number of
particles released per infected bacteria may be as high as 1000.
Assay for Lytic Phage
a. Plaque assay - Lytic phage are enumerated by a plaque assay. A plaque is a clear area
which results from the lysis of bacteria. Each plaque arises from a single infectious phage. The
infectious particle that gives rise to a plaque is called a pfu (plaque forming unit).

B. Lysogenic or Temperate Phage

1. Definition - Lysogenic or temperate phages are those that can either multiply via the lytic
cycle or enter a quiescent state in the cell. In this quiescent state most of the phage genes are
not transcribed; the phage genome exists in a repressed state. The phage DNA in this
repressed state is called a prophage because it is not a phage but it has the potential to
produce phage. In most cases the phage DNA actually integrates into the host chromosome
and is replicated along with the host chromosome and passed on to the daughter cells. The cell
harboring a prophage is not adversely affected by the presence of the prophage and the
lysogenic state may persist indefinitely. The cell harboring a prophage is termed a lysogen.
2. Events Leading to Lysogeny - The Prototype Phage: Lambda
a. Circularization of the phage chromosome - Lambda DNA is a double stranded linear
molecule with small single stranded regions at the 5' ends. These single stranded ends are
complementary (cohesive ends) so that they can base pair and produce a circular molecule. In
the cell the free ends of the circle can be ligated to form a covalently closed circle as illustrated
in Figure 5.
b. Site-specific recombination - A recombination event, catalyzed by a phage coded enzyme,
occurs between a particular site on the circularized phage DNA and a particular site on the host
chromosome. The result is the integration of the phage DNA into the host chromosome as
illustrated in Figure 6.
c. Repression of the phage genome - A phage coded protein, called a repressor, is made
which binds to a particular site on the phage DNA, called the
operator, and shuts off transcription of most phage genes
EXCEPT the repressor gene. The result is a stable
repressed phage genome which is integrated into the host
chromosome. Each temperate phage will only repress its
own DNA and not that from other phage, so that repression
is very specific (immunity to superinfection with the same
phage).
3. Events Leading to Termination of Lysogeny
Anytime a lysogenic bacterium is exposed to adverse
conditions, the lysogenic state can be terminated. This
process is called induction. Conditions which favor the
termination of the lysogenic state include: desiccation,
exposure to UV or ionizing radiation, exposure to mutagenic
chemicals, etc. Adverse conditions lead to the production of
proteases (rec A protein) which destroy the repressor
protein. This in turn leads to the expression of the phage
genes, reversal of the integration process and lytic
multiplication.
4. Lytic vs Lysogenic Cycle
The decision for lambda to enter the lytic or lysogenic cycle when it first enters a cell is
determined by the concentration of the repressor and another phage protein called cro in the
cell. The cro protein turns off the synthesis of the repressor and thus prevents the establishment
of lysogeny. Environmental conditions that favor the production of cro will lead to the lytic cycle
while those that favor the production of the repressor will favor lysogeny.

5. Significance of Lysogeny
a. Model for animal virus transformation - Lysogeny is a model system for virus
transformation of animal cells
b. Lysogenic conversion - When a cell becomes lysogenized, occasionally extra genes
carried by the phage get expressed in the cell. These genes can change the properties of the
bacterial cell. This process is called lysogenic or phage conversion. This can be of significance
clinically. e.g. Lysogenic phages have been shown to carry genes that can modify the
Salmonella O antigen, which is one of the major antigens to which the immune response is
directed. Toxin production by Corynebacterium diphtheriae is mediated by a gene carried by a
phage. Only those strain that have been converted by lysogeny are pathogenic.
 LYTIC AND LYSOGENIC CYCLES - PHAGE MULTIPLICATION CYCLE

A. Definition - Lytic or virulent phages are phages which can only multiply on bacteria and kill
the cell by lysis at the end of the life cycle.

Lytic or Virulent Phages

a. Eclipse period - During the eclipse phase, no infectious phage particles can be found either
inside or outside the bacterial cell. The phage nucleic acid takes over the host biosynthetic
machinery and phage specified m-RNA's and proteins are made. There is an orderly expression
of phage directed macromolecular synthesis, just as one sees in animal virus infections. Early
m-RNA's code for early proteins which are needed for phage DNA synthesis and for shutting off
host DNA, RNA and protein biosynthesis. In some cases the early proteins actually degrade the
host chromosome. After phage DNA is made late m-RNA's and late proteins are made. The late
proteins are the structural proteins that comprise the phage as well as the proteins needed for
lysis of the bacterial cell.
b. Intracellular Accumulation Phase - In this phase the nucleic acid and structural proteins
that have been made are assembled and infectious phage particles accumulate within the cell.
c. Lysis and Release Phase - After a while the bacteria begin to lyse due to the accumulation
of the phage lysis protein and intracellular phage are released into the medium. The number of
particles released per infected bacteria may be as high as 1000.

Assay for Lytic Phage

a. Plaque assay - Lytic phage are enumerated by a plaque assay. A plaque is a clear area
which results from the lysis of bacteria. Each plaque arises from a single infectious phage. The
infectious particle that gives rise to a plaque is called a pfu (plaque forming unit).

B. Lysogenic or Temperate Phage

1. Definition - Lysogenic or temperate phages are those that can either multiply via the lytic
cycle or enter a quiescent state in the cell. In this quiescent state most of the phage genes are
not transcribed; the phage genome exists in a repressed state. The phage DNA in this
repressed state is called a prophage because it is not a phage but it has the potential to
produce phage. In most cases the phage DNA actually integrates into the host chromosome
and is replicated along with the host chromosome and passed on to the daughter cells. The cell
harboring a prophage is not adversely affected by the presence of the prophage and the
lysogenic state may persist indefinitely. The cell harboring a prophage is termed a lysogen.
2. Events Leading to Lysogeny - The Prototype Phage: Lambda
a. Circularization of the phage chromosome - Lambda DNA is a double stranded linear
molecule with small single stranded regions at the 5' ends. These single stranded ends are
complementary (cohesive ends) so that they can base pair and produce a circular molecule. In
the cell the free ends of the circle can be ligated to form a covalently closed circle as illustrated
in Figure 5.
b. Site-specific recombination - A recombination event, catalyzed by a phage coded enzyme,
occurs between a particular site on the circularized phage DNA and a particular site on the host
chromosome. The result is the integration of the phage DNA into the host chromosome as
illustrated in Figure 6.
c. Repression of the phage genome - A phage coded protein, called a repressor, is made
which binds to a particular site on the phage DNA, called the operator, and shuts off
transcription of most phage genes EXCEPT the repressor gene. The result is a stable repressed
phage genome which is integrated into the host chromosome. Each temperate phage will only
repress its own DNA and not that from other phage, so that repression is very specific (immunity
to superinfection with the same phage).
3. Events Leading to Termination of Lysogeny
Anytime a lysogenic bacterium is exposed to adverse conditions, the lysogenic state can be
terminated. This process is called induction. Conditions which favor the termination of the
lysogenic state include: desiccation, exposure to UV or ionizing radiation, exposure to
mutagenic chemicals, etc. Adverse conditions lead to the production of proteases (rec A protein)
which destroy the repressor protein. This in turn leads to the expression of the phage genes,
reversal of the integration process and lytic multiplication.
4. Lytic vs Lysogenic Cycle
The decision for lambda to enter the lytic or lysogenic cycle when it first enters a cell is
determined by the concentration of the repressor and another phage protein called cro in the
cell. The cro protein turns off the synthesis of the repressor and thus prevents the establishment
of lysogeny. Environmental conditions that favor the production of cro will lead to the lytic cycle
while those that favor the production of the repressor will favor lysogeny.

5. Significance of Lysogeny
a. Model for animal virus transformation - Lysogeny is a model system for virus
transformation of animal cells
b. Lysogenic conversion - When a cell becomes lysogenized, occasionally extra genes
carried by the phage get expressed in the cell. These genes can change the properties of the
bacterial cell. This process is called lysogenic or phage conversion. This can be of significance
clinically. e.g. Lysogenic phages have been shown to carry genes that can modify the
Salmonella O antigen, which is one of the major antigens to which the immune response is
directed. Toxin production by Corynebacterium diphtheriae is mediated by a gene carried by a
phage. Only those strain that have been converted by lysogeny are pathogenic.
 VIROIDS, PRIONS

A virus is a small infectious agent that can replicate only inside the living cells of
organisms. Most viruses are too small to be seen directly with a light microscope. Viruses infect
all types of organisms, from animals and plants to bacteria and archaea. Since the initial
discovery of tobacco mosaic virus by Martinus Beijerinck in 1898, about 5,000 viruses have
been described in detail though there are millions of different types. Viruses are found in almost
every ecosystem on Earth and are the most abundant type of biological entity. The study of
viruses is known as virology, a sub-speciality of microbiology.
Virus particles (known as virions) consist of two or three parts: the genetic material made
from either DNA or RNA, long molecules that carry genetic information; a protein coat that
protects these genes; and in some cases an envelope of lipids that surrounds the protein coat
when they are outside a cell. The shapes of viruses range from simple helical and icosahedral
forms to more complex structures. The average virus is about one one-hundredth the size of the
average bacterium.
• Every virus has 2 stages
dormant, particulate, transmissible stage called the virion stage
an active, intracellular stage called the infectious stage
Virion Stage
• Virions are the transmissible state of a virus. Metabolically inert.
• Virion = "a piece of nucleic acid wrapped up in a protein coat" (and/or a membrane)
• The nucleic acid can be either DNA (double-stranded (ds) or single-stranded (ss)) or RNA (ds
or ss); never both.
• The coat (also called viral shell or capsid) can be icosahedron (20-sided regular geometric
shape common in many bacterial, animal, and plant viruses), sphere, cylinder, bullet-
shaped, or amorphous shaped particle.
• Virions must be able to adhere and allow entry into some host cell(s). Also to survive outside
of host cell environment.
• Some virions more hardy than others (e.g., hepatitis virus can withstand short periods of
boiling; most virions are destroyed by this).

Infectious Stage
• When virus infects a cell, nucleic acid must be uncoated and gain access to metabolic
machinery of cell.
• Virus life cycle is characterized by:
attachment
penetration, with entry of nucleic acid into cell
early expression of virus genes (either directly by translation, if virus contains "+" RNA,
or indirectly after transription and then translation)
replication of virus nucleic acid
synthesis of new virion components
packaging and assembly of new virions
exit from cell

Measurement of viral growth

• Must grow virus on host cells to see anything. Can't grow virus without cells.
• To quantify viruses, need some way to get flat surface of growing cells, allow virus-infected
cells to spread radially where present = plaque.
• In bacterial cells this is easy. Spread "lawn" of bacteria on plate, add diluted phage
suspension or culture infected with phages. After 6-8 hours can see plaques in E. coli.
• In plant cells, can be easy. Example: Tobacco Mosaic Virus (TMV), make virus dilution, rub
over surface of tobacco leaf. After leaf growth, can observe plaque areas.
• In animal cells, not so easy. In 1960's, standard assay was to inoculate chicken egg
membranes of developing chick embryos, incubate for a week, cut open shell and count
plaques on membrane in the air sac. Lots of work to get statistically reliable data!
• In 1970's tissue culture became a viable alternative. Animal cells are cultured as microbes in
glass or plastic, use special medium that contains most of nutrients present in blood.
Cells will spread as monolayer on surface, can count plaques after staining.

Taxonomy of viruses
• Based mainly on Virion and Kingdom of host
• Use Host cell type (Animal viruses, plant viruses, etc.)
• Use Nucleic Acid type (ds DNA, ss DNA, ds RNA, ss RNA)
• Use + or - polarity of RNA. "+" is able to serve as mRNA. "-" is the complement of +, must
function as template to make a complementary strand of + RNA before any translation
can occur.
• Use virus coat morphology. Enveloped vs. non-enveloped viruses.
Virion Structure
"Naked" viruses
1. Helical viruses
Tobacco mosaic virus (TMV) is an example of a virus with helical symmetry.
A helical array of identical protein subunits surrounds an RNA molecule.

2. Icosahedral viruses
built from icosahedral (20-sided) assemblies of protein subunits.
Icosahedral shape is the minimum free energy structure for producing a shell
of equivalently bonded identical structures.
The simplest icosahedral capsids are built up by using 3 identical subunits to
form each triangular face, thereby requiring 60 identical subunits to form a
complete capsid. A few simple virus particles are constructed in this way,
e.g. bacteriophage ØX174.
Most icosahedral viruses have more than 60 subunits, usually some multiple N
times 60. N (called the triangulation number) can have values of 1, 3, 4,
7, 9, 12, and more.
"Enveloped" viruses
3. "Naked" viruses require host death so viruses can be released. This may be wasteful,
and may cause premature death of host cell.
4. Alternative strategy: shed virus particles by budding out, continued release from cell
membrane. Cell does not die (immediately), continues to serve as factory for
virus assembly and release. Virus typically acquires a coating of host cell
membrane, modified to include virus-specific proteins. This is the "envelope".
Virus may have additional protein coats (often icosahedral) inside the envelope.
5. Eventually host cell is too depleted to survive. Can see evidence of this as "cytopathic
effect" (CPE). Cell then dies.
6. Examples of enveloped viruses include:
Retrovirus, including HIV
Paramyxovirus, including influenza
Rhabdovirus, including rabies
Filovirus. Although very "hot" in the news, these viruses are very poorly
characterized because of their extreme pathogenicity. They are class IV
pathogens, meaning they can only be cultured in total containment
facilities, of which there are only two in the U. S. They are thought to be
enveloped viruses with - RNA genomes.

Virus Genomes
• Rule of Thumb: to estimate # of virus proteins, look at size of viral DNA or RNA. For each
1000 base pairs, can guess the existence of 1 protein
1. "typical" gene has 300-400 amino acids = ~ 1000 base pairs = 1 kbp (= 1 protein)
2. small virus: SV40 => 5000 base pairs = 5 kbp ~ 5 proteins
3. large virus: T4 => 200 kbp ~ 100-200 proteins
4. by comparison, E. coli: 4000 kbp

Bacterial Viruses = Phages

Bacterial defenses against infection
Cell surfaces: possibilities of mutation
• Virus must attach to some specific cell surface protein or polysaccharide. But these are
specified by genes, and genes can mutate. In population, will always find some variant
strains with slightly different cell surfaces, may not bind virus well.
 • When phage first discovered, thought this could be effective weapon against bacterial
disease. But frequency of resistant bacterial strains was too high, any given strain of
virus quickly became useless as resistant survivors propagated.

Nucleases: endo- and exo-DNases and RNases

• All bacteria seem to have nucleases that can attack DNA (called DNases) and RNA
(called RNases).
• Exoenzymes attack free 5' or 3' ends of DNA, RNA molecules. Bacteria are protected
since DNA (and plasmids) are always circular. RNases are present, and in fact destroy
mRNA eventually (bacteria are always making new RNAs, very responsive to
enviroment changes).
• Endonucleases are potentially lethal weapons. Called restriction enzymes. Attack at
specific sequence: e.g., in E. coli, enzyme called EcoRI will attack any sequence with 5'
G-A-A-T-T-C 3' (cuts DNA between G and A).
• Why doesn't this kill cell? Because cell also has a second enzyme, called modification
enzyme, that protects all host DNA sequences of this type. Typically adds a methyl (-
CH3) group to one base at the cutting site. The methylated base is modified, and
protected from the restriction enzyme. When foreign DNA comes into cell (e.g. virus
DNA), if restriction site if present it will be cut and ----- requiem for the virus.

The importance of Restriction Enzymes

Restriction enzymes are responsible for the genetic revolution. They make reproducible, specific
cuts with surgical precision. Major industry has emerged in biochemical supply companies to
harvest bacteria, purify restriction enzymes, and sell these to research and applied industries.

Animal Viruses
• Animal viruses are different in many respects from bacterial viruses. The host cells are more
complex, with multiple compartments and more complex regulation of replication,
transcription, and translation. Animal cells are not bounded by cell walls.
• Not surprisingly, animal viruses have evolved to overcome these problems. They attach and
enter by different mechanisms than phages, and their intracellular activities include the
ability to move between different compartments as needed.
• Viral entry and exit from cells is very different from bacteriophages. Animal viruses must enter
through cell membrane, either by triggering endocytosis pathway or by fusing viral
 envelope with the cell envelope.
• Modifications are needed in both cellular and viral mRNA to allow recognition and movement
from nucleus to cytoplasm. For example:
1. 3' tail of poly A
2. 5' cap of methyl Guanosine triphosphate

Types of infection
Viruses in animal cells show a variety of infection patterns:
• lytic infection: destroys host cells.
• persistent infection: host cell continues to shed virus over long time. Cell gradually becomes
recognizably poorer (recognized as cytopathic effect, or CPE), eventually "crumps out".
• transformation: infection by certain viruses causes cells to change, become cancerous.
Responsible genes are called oncogenes (tumor-producing genes). Viral oncogenes
have also been found in uninfected cells. These are genes involved in regulation of cell
 cycle; when defective, normal regulatory control is lost and cell can become cancerous.
• latent infection: virus genes may not be expressed for long time (ex. many Herpes
infections). Not the same as lysogeny -- genes are not integrated into host chromosome.

Human Immunodefiency Virus (HIV) and AIDS

1. AIDS first recognized in 1981. Over 300,000 cases reported in U.S., over 8 million in Africa,
over 12 million infected world-wide.

2. View AIDS in perspective: a PBS website with current data on the AIDS epidemic.

3. Transmission: sexual activity, especially with multiple sex partners. Also contaminated blood,
needles, hospitals. Not just a disease of homosexuals! In Africa (most # cases) about
equal # of male and female victims.

4. AIDS lowers immune system's ability to respond to other infections, allows opportunistic
pathogens to invade body. Most common infection is pneumonia (lung infection) caused
by Pneumocystis (2/3 of all AIDS patients get this at some point).

5. Host cell for the virus is CD4 (T-helper) cell, needed to activate antibody production. In normal
human, CD4 cells account for 70% of total T cells -- in AIDS, number decreases, may
reach 0% of T cell pool.

6. Progress of HIV infection only recently understood. Formerly thought that virus became latent.
Now discover that virus is anythig but latent: during infected period (which can last 10
years), body is destroying ~ billion virions/day, and virus is killing about 100 million CD4
T cells a day. HIV virus continues replicating, and body rapidly replenishes lost T cells.
Only when lymph nodes wear out does virus gain the upper hand. See handout in class
titled "Huge HIV turnover helps explain drug resistance, pathogenicity".

7. Prognosis: with carefully selected treatments, better than before. Virtually every infected
person dies sooner of later, usually within 10 years of infection. No cure known, no
vaccine yet available. Virus mutates rapidly, many strain variations. Vaccines being tried,
results mixed but preliminary.
8. Drugs: some types of drugs offer limited success.
1. AZT (azidothymidine) is analog of thymidine, but is blocked at 3'-position, so no further
chain growth possible. These target viral reverse transcriptase enzyme. Should
reduce DNA synthesis in treated cells. But eventually, viral mutants resistant to
drug arise. Also, long term use of drug can cause toxic side effects.
2. protease inhibitors. Like many viruses, HIV needs to cleave large protein product into
smaller products, using viral protease protein. By inhibiting this enzyme, should
block necessary stage in viral replication cycle. Still under development, but
resistant viral mutants
to these type of drugs
have already been
found. Still, drug offers
promise. See handout
article for more details.
Safe sex! Caution with
sharps. Extra caution
in clinical settings.

HIV Life Cycle

(budding through plasma
membrane)
Viroids and Prions
• Viroids very small ss RNA genomes (~300 nucleotides). No coat, and RNA does not encode
protein. Known viroids cause diseases in plants because host cells replicate the RNA.
• Prions (protein infectious agent) do not have a nucleic acid genome. Prion diseases are often
called spongiform encephalopathies because of the post mortem appearance of the
brain with large vacuoles in the cortex and cerebellum.
• Pathology of brains infected with prion diseases:
1. Scrapie (sheep)
2. bovine spongiform encephalopathy (cows) = "mad cow disease"
3. Creutzfeldt-Jakob Disease (humans)
• Prion diseases in humans are probably primarily a genetic neurotoxic disorder. Transmission
of the disease to humans via infectious prions is likely to be rare.
• The prion is a modified form of a normal cellular protein known as PrPc (for cellular), found
predominantly on the surface of neurons and thought to be involved in synaptic function.
The modified form of PrPc (= prion) is known as PrPsc (for scrapie) which is relatively resistant
to proteases and accumulates in cytoplasmic vesicles of diseased individuals. Prion protein may
cause normal protein to fold abnormally.
 BACTERIAL GENETICS

Bacterial genetics is the study of gene structure and function in bacteria. Genetics
itself is concerned with determining the number, location, and character of the genes of an
organism. The process of replication is essential to understand in or manipulate the genome
and to understand the functioning of these organisms.
DNA REPLICATION
In general, DNA is replicated by uncoiling of the helix, strand separation by breaking of the
hydrogen bonds between the complementary strands, and synthesis of two new strands by
complementary base pairing. Replication begins at a specific site in the DNA called the origin
of replication.

How does the DNA in the bacterial cell replicate ……

Transcription in bacteria… how does it happen……

Transcription is the process by which genetic information from DNA is transferred

into RNA. DNA sequence is enzymatically copied by RNA polymerase to produce a
complementary nucleotide RNA strand. One significant difference between RNA and DNA
sequence is the presence of U, or uracil in RNA instead of the T, or thymine of DNA. In the
case of protein-encoding DNA, transcription is the first step that ultimately leads to the
translation of the genetic code, via the mRNA intermediate, into a functional peptide or
protein. The stretch of DNA that is transcribed into an RNA molecule is called a transcription
unit. A transcription unit that is translated into protein contains sequence that directs and
regulates protein synthesis in addition to coding sequence that is translated into protein.
Regulatory sequence that is before, or 5', of the coding sequence is called 5' untranslated
(5'UTR) sequence, and sequence found following, or 3', of the coding sequence is called 3'
untranslated (3'UTR) sequence. As in DNA replication, transcription proceeds in the 5' → 3'
direction. The DNA template strand is read 3' → 5' by RNA polymerase and the new RNA
strand is synthesized in the 5'→ 3' direction. RNA polymerase binds to the 3' end of a gene
(promoter) on the DNA template strand and travels toward the 5' end. Except for the fact
that thymines in DNA are converted to uracils in RNA, the newly synthesized RNA strand will
have the same sequence as the coding (non-template) strand of the DNA.

Prokaryote Eukaryote
How to undertake experiments for understanding genetics of bacteria ?

The classical way to investigate genes is to mate two organisms with different
genotypes and compare the observable properties (phenotypes) of the parents with those of
the progeny. Bacteria do not mate (in the usual way), so there is no way of getting all the
chromosomes of two different bacteria into the same cell. However, there are a number of
ways in which a part of the chromosome or genome from one bacterium can be inserted
into another bacterium so that the outcome can be studied.
The first step in performing genetic research on bacteria is to select mutants that
differ from wild-type cells in one or more genes. Then crosses are made between mutants
and wild types, or between two different mutants, to determine dominance-recessive
relationships, chromosomal location, and other properties. Various genetic methods are
used to select bacterial mutants, antibiotic-resistant cells, cells with specific growth
requirements, and so on.
• Mutants in bacteria are mostly biochemical in nature, because we can’t generally see
the cells.
• The most important mutants are auxotrophs. An auxotroph needs some nutrient
that the wild type strain (prototroph) can make for itself. For example, a trp-
auxotroph can’t make its own tryptophan (an amino acid). To grow trp- bacteria,
you need to add tryptophan to the growth medium. Prototrophs are trp+; they don’t
need any tryptophan supplied since they make their own.
• Chemoauxotrophs are mutants that can’t use some nutrient (usually a sugar) that
prototrophs can use as food. For example, lac- mutants can’t grow on lactose (milk
sugar), but lac+ prototrophs can grow on lactose.
• Resistance mutants confer resistance to some environmental toxin: drugs, heavy
metals, bacteriophages, etc. For instance, AmpR causes bacteria to be resistant to
ampicillin, a common antibiotic related to penicillin.
• Auxotrophs and chemoauxotrophs are usually recessive; drug resistance mutants are
usually dominant. A common way to find bacterial mutants is replica plating, which
means making two identical copies of the colonies on a petri plate under different
conditions.
• For instance, if you were looking for trp- auxotrophs, one plate would contain added
tryptophan and the other plate would not have any tryptophan in it.
• Bacteria are first spread on the permissive plate, the plate that allows both mutants
and wild type to grow, the plate containing tryptophan in this case. They are allowed
 to grow fro a while, then a copy of the plate is made by pressing a piece of velvet
onto the surface of the plate, then moving it to a fresh plate with the restrictive
condition (no tryptophan). The velvet transfers some cells from each colony to an
identical position on the restrictive plate.
• Colonies that grow on the permissive plate but not the restrictive plate are
(probably) trp- auxotrophs, because they can only grow if tryptophan is supplied.

Replica plating
The Ames test uses bacteria to test chemicalsfor capacity to cause mutations, as well as
carcinogens (cancer-causing chemicals). The procedure is as fiollows:
 GENE EXPRESSION
Is the process by which information from a gene is used in the synthesis of a
functional gene product. These products are often proteins, but in non-protein coding genes
such as rRNA genes or tRNA genes, the product is a functional RNA. The process of gene
expression is used by all known life - eukaryotes (including multicellular organisms),
prokaryotes (bacteria and archaea) and viruses - to generate the macromolecular
machinery for life. Several steps in the gene expression process may be modulated,
including the transcription, RNA splicing, translation, and post-translational modification of a
protein. Gene regulation gives the cell control over structure and function, and is the basis
for cellular differentiation, morphogenesis and the versatility and adaptability of any
organism. Gene regulation may also serve as a substrate for evolutionary change, since
control of the timing, location, and amount of gene expression can have a profound effect
on the functions (actions) of the gene in a cell or in a multicellular organism.
In genetics, gene expression is the most fundamental level at which genotype gives rise to
the phenotype. The genetic code is "interpreted" by gene expression, and the properties of
the expression products give rise to the organism's phenotype.

Transcription (Video)
The gene itself is typically a long stretch of DNA. It is a blueprint for the production of RNA.
The production of RNA copies of the DNA is called transcription, and is performed by RNA
polymerase, which adds one RNA nucleotide at a time to a growing RNA strand. This RNA is
complementary to the template 3' → 5' DNA strand,[1] which is itself complementary to the
coding 5' → 3' DNA strand. Therefore, the resulting 5' → 3' RNA strand is identical to the
coding DNA strand with the exception that thymines are replaced with uracils in the RNA. A
coding DNA strand reading "ATG" is transcribed as "UAC" in RNA.
Translation (Video)
• Initiation
• 30S initiates binding to mRNA.
• locates Shine-Dalgarno sequence (3-9 bases near 5' end of mRNA).
• ribosome finds first AUG codon.
• 50S ribosome binds.
• tRNA carries N-formylmethione to first position
• Elongation
• 2 adjacent sites on ribosome: P and A site.A site accepts a new tRNA-AA.Psite holds
existing chain peptide transferred from P site tRNA to A-site AA
• enzyme activity is in ribosomal RNA, not protein
• also required: Energy (GTP) and elongation factors
• Termination
• reach a "stop codon" UAG, UAA, or UGA
• no t-RNAs for release, but release factors required
• Net cost: 4 phosphate bonds/amino acid added!
• B. Genetic Code
1. AUG = universal "start" codon
2. UAG, UAA, UGA = "stop" codons
3. A few messages in bacteria use GUG as start, but still need Shine-Dalgarno
sequence, stil code for N-formylmethionine.
 Classic example: The lac operon (Video)

• gene is regulated by negative control; in absence of specific repressor, gene is transcribed

just like constitutive gene. In order to regulate, must add specific block. Must say
"no"; otherwise gene is not down-regulated.
• Lactose = milk sugar, disaccharide made of galactose + glucose. In order to metabolize
lactose, cells must produce enzyme ß-galactosidase, split lactose into galactose +
glucose
• Observation: add lactose to cells: within minutes, ß-galactosidase enzyme appears, also
lac permease in membrane, and a third protein, transacetylase. Level of ß-
galactosidase enzyme can accumulate to level of 10% of cytoplasmic protein
• Explanation: in absence of lactose (= inducer), lac repressor blocked operator site.
• Lac repressor is allosteric protein. Coded for by another region of DNA (constitutive gene,
weak promoter, low level of expression)
• Effector molecule = Inducer is lactose (actually allolactose, or analog such as IPTG) binds
to repressor protein, repressor released, RNA is made, all genes turned on as unit
Some genes are regulated by activator proteins
• genes with weak promoters are rarely transcribed.some such genes can be activated by
activator protein, causes RNA polymerase to bind more tightly.often there are two
components to such regulation: a sensor protein and an activator protein. The
activator protein is inactive until phosphorylated by the sensor, then it activates
transcription, gene product is made.
Lac operon – in presence of inducer ( lactose)
 Lac operon – in absence of lactose

Role of mRNA
Carries coding information for amino acids = codons, 3 adajacent nucleotide bases
Example: AAA, AGU, etc.leader sequence on mRNA (called Shine-Dalgarno sequence) binds
to complementar sequence on small ribosome subunit.
 

Role of ribosome
acts as a "decoding box" or "tape player" for the information in mRNA30S & 50S subunits
(= 70S)30S has 16S RNA + 21 proteins.50S has 23S & 5S RNA + 34 proteins.
Role of tRNA
4. structure: 4 loops, anticodon, AA binding site
5. ~ 60 types in bacteria (>100 in mammals)
6. only 73-93 nucleotides long
7. some modified bases: pseudouridine, inosine, others
8. modified after transcription
9. extensive hairpin loops
10. anticodon site: recognizes codon on mRNA
11. AA added by enzyme: AA-tRNA activating enzymes
12. ATP required, forms AA-AMP + PP, then AA-tRNA + AMP
 RECOMBINATION IN BACTERIA
Transfer of Genetic Material in Bacteria
The process of transfer of genetic material and recombination is very interesting
bascterial recombination is given (PPT. an overview of bacterial recombination).The
three main mechanisms by which bacteria acquire new DNA are transformation,
conjugation, and transduction. Transformation involves acquisition of DNA from the
environment, conjugation involves acquisition of DNA directly from another bacterium, and
transduction involves acquisition of bacterial DNA via a bacteriophage intermediate.
Transformation
Transformation is the process by which bacteria pick up DNA from their environment.
The DNA may come from a variety of sources, but most likely it is the remnants of DNA
from dead bacterial cells.
In order to become successfully transformed, bacteria must be competent. This means
that the bacteria are expressing the appropriate enzymes (the 'transformation machinery')
required to transport the exogenous DNA into the cell. Therefore, the correct genes must be
expressed in order to carry out transformation. Expression of these genes depends on the
growth conditions: bacteria most likely to be competent are dividing rapidly, but nutrients in
the environment are becoming limited. (For more on the control of gene expression, see the
module on bacterial gene regulation.
In transformation, a cell surface receptor binds to DNA in the environment. After
binding, the DNA is transported across the membrane by the transformation machinery. As
this occurs, one strand of the DNA is digested away by an exonuclease, so that the DNA
that enters the cell is single stranded. This promotes recombination, as long as the DNA
taken up is sufficiently homologous to the host DNA to allow recombination to occur. The
recombination that occurs is one-way (non-reciprocal); unlike the exchange of strands
diagrammed in the module on recombination, in this case the new DNA will simply replace a
strand of the host DNA. The replaced segment of host DNA will be degraded. If the new
DNA is of a different allelic nature than the host DNA, a gene conversion event can occur.
This is what happened in the example mentioned above: the avirulent strain of S.
pneumoniae had a mutation in a gene required for production of the bacterial capsule. Heat
killing the virulent cells (which contained the wild-type capsule gene) caused the release of
fragments of the dead cells' genomes. Some of the avirulent cells picked up a piece of DNA
containing the wild-type capsule gene, and underwent gene conversion so that they were
wild type for that gene, causing them to become virulent.
Conjugation
Conjugation is a mating process involving bacteria. It involves transfer of genetic
information from one bacterial cell to another, and requires physical contact between the
two bacteria involved. The contact between the cells is via a protein tube called an F or sex
pilus, which is also the conduit for the transfer of the genetic material.
Basic conjugation involves two strains of bacteria: F+ and F-. The difference
between these two strains is the presence of a Fertility factor (or F factor) in the F+ cells.
The F factor is an episome that contains 19 genes and confers the ability to conjugate upon
its host cell. Genetic transfer in conjugation is from an F+ cell to an F- cell, and the genetic
material transferred is the F factor itself. Here is an overview of the process:
Basic conjugation occurs between an F+ cell and an F- cell.
The difference between these two types of cells is the
presence or absence of the F (fertility) factor, which is a
circular DNA molecule independent of the bacterial
chromosome (the larger circular molecule.

The F+ cell initiates conjugation by extending an F pilus

toward the F- cell. Among the genes present on the F factor
are the genes encoding the proteins required for pilus
construction.

The F pilus, when finished, temporarily connects the two

cells. On strand of the F factor is nicked, and begins
unwinding from the other strand. The nicked strand begins
to transfer through the F pilus to the F- cell. As it does so,
this strand begins to be replicated, as does circular strand
remaining behind in the F+ cell.

Eventually, the nicked strand completely passes through to

the recipient cell, and is completely replicated. This process
produces a new F factor in the recipient cell. The pilus is
broken, severing the connection between the two cells.
Since both cells now contain an F factor, both cells are F+.
The new F+ cell (which was the F- cell, can now initiate
conjugation with another F- cell.

Recombination rarely occurs with this kind of conjugation. This is because the F
factor is not homologous to the DNA in the bacterial chromosome. As we will see, however,
there are variations of this basic conjugation process that allow recombination to occur.
Conjugation Involving Hfr Bacteria
Occasionally, the F factor integrates into a random position in the bacterial
chromosome. When this happens, the bacterial cell is called Hfr instead of F+. Hfr bacteria
are still able to initiate conjugation with F- cells, but the outcome is completely different
from conjugation involving F+ bacteria:
As mentioned above, Hfr cells are formed when the F factor
integrates into the bacterial chromosome. This integration occurs
at a random location.
The Hfr cell is still able to initiate conjugation with an F- cell.
When DNA transfer begins, the Hfr cell tries to transfer the entire
bacterial chromosome to the F- cell. The first DNA to be
transferred is chromosomal DNA, and the last DNA to be
transferred will be the F factor DNA.
Transfer of the bacterial chromosome is almost never complete.
Pili are fairly fragile structures, and shear forces tend to break the
pilus, disrupting DNA transfer before the entire chromosome can
be transferred. As a result, the F factor itself is almost never
transferred to the recipient cell. This cell will remain F-. This cell
will receive new DNA from the Hfr cell however, and this new DNA
can undergo recombination at a high frequency with the host
chromosome, because the DNA sequences will be homologous. In
fact, Hfr is short for 'high frequency recombination'. This
recombination can result in gene conversion events, if the
transferred DNA and the corresponding region of host DNA contain
different alleles of the same gene.

Mapping Genes on Bacterial Chromosomes

Bacteria, since they are usually haploid, cannot have their chromosomes mapped by
the same techniques as eukaryotes (For a reminder of how this works, see the module on
linkage and mapping). They can, however, be mapped by using Hfr bacterial conjugation.
For example, imagine that an F- cell has mutant alleles of two genes, a and b (the F- would
therefore be a-, b-). If this cell undergoes conjugation with an Hfr cell that is a+, b+ (in
other words, wild type), the F- cell should undergo gene conversion to a+, b+ when both of
those genes have been transferred by conjugation. By determining how long it takes the b
gene to transfer after the a gene has transferred, it is possible to get a relative idea of how
far apart the two genes are on a chromosome.
The experiment would be done this way: a+, b+ Hfr cells would be mixed with a-,b-
F- cells. The time of mixing would be designated 'time zero'. At regular intervals, a small
amount of the mixture would be removed and conjugation would be disrupted using a
blender (the shear force of the blender would cause any pili to break). These bacteria would
then be tested for gene conversion (for example, if the mutations rendered the F- bacteria
auxotrophic, the bacteria could be tested by growing them on minimal medium, or minimal
medium supplemented with the necessary nutrient required because of one or the other
mutation). If the a gene was converted to wild type at 8 minutes after time zero, and the b
gene was converted to wild type at 19 minutes after time zero, then the distance between
the two genes would be '11 minutes' (because that was the difference in time required to
transfer the b gene compared to the a gene). Bacterial map distances are always expressed
in minutes, because of this technique.
F' Conjugation
Just as F factors can occasionally integrate into the bacterial chromosome (producing
an Hfr cell from an F+ cell), integrated F factors can occasionally excise themselves from
the bacterial chromosome. If this excision occurs properly, the Hfr cell becomes an F+
again. The excision is sometimes sloppy, however, and the F factor takes a small segment
of the bacterial chromosome with it. Some of the chromosomal DNA has therefore become
associated with the episome. When this happens, the cell is called an F'.
Conjugation involving F' cells allows for the possibility of recombination, as shown below:
The F' cell has a full complement of chromosomal genes; however, some of those genes
are now on the episome. F' cells are able to initiate conjugation with F- cells because of
the presence of the F factor.
When the F factor begins to transfer its DNA to the recipient cell, it will transfer the small
segment of chromosomal DNA as well.
Just as in the F+/F- mating, both cells wind up with a copy of the episome. The cell that
was F- now has the F factor (along with the piece of chromosomal DNA) and is therefore
now F'. This cell, however, also has a complete chromosome, so it will be diploid for the
segment of chromosomal DNA on the episome. Such a partially diploid bacterial cell is
called a merozygote. The chromosomal DNA on the episome can undergo recombination
at high frequency with its homologous sequence on the chromosome.

Transduction
Transduction involves the exchange of DNA between bacteria using bacterial viruses
(bacteriophage) as an intermediate. There are two types of transduction, generalized
transduction and specialized transduction, which differ in their mechanism and in the DNA
that gets transferred. Before we can address these processes, however, we need to
understand the life cycle of a bacteriophage.
When a phage infects a bacterial cell, it injects its DNA into the cell. The viral DNA is
replicated numerous times, and viral genes are expressed, producing the proteins that
make up the viral capsid (or protein coat) and nucleases that digest the host genome into
fragments. The newly replicated viral DNA molecules are packaged into viral capsids, and
the bacterial cell is lysed (burst, and therefore killed), releasing hundreds of viral progeny,
which then go on to infect other cells.
Generalized Transduction
Sometimes, during bacteriophage replication, a mistake is made, and a fragment of
the host DNA gets packaged into a viral capsid. The resulting phage would be able to infect
another cell, but it would not have any viral genes, so it would not be able to replicate. The
cell infected by this phage would survive, and would have an extra piece of bacterial DNA
present, which could undergo recombination with the host chromosome, and perhaps cause
a gene conversion event. Because it is a random fragment that gets packaged into the viral
capsid, any segment of the bacterial DNA can be transferred this way (hence the name
'generalized').
Specialized Transduction
Specialized transduction occurs only with certain types of bacteriophage, such as
phage lambda. Lambda has the ability to establish what is called a lysogenic infection in
a bacterial cell. In a lysogenic infection, the viral DNA becomes incorporated into the host
chromosome, much as the F factor did in Hfr cells. In a lysogenic infection by lambda, the
DNA integrates into a very specific spot in the host chromosome. The integrated viral DNA
can remain integrated for long periods of time, without disturbing the cell. Under the
appropriate conditions (the regulation of this is very complex, so don't worry about it), the
viral DNA will excise itself from the chromosome, and enter the lytic phase, in which the
virus replicates just as described above. The cell gets lysed, and new bacteriophage
particles are released to infect other cells. As with excision of the F factor (when Hfr cells
become F'), sometimes the excision of lambda is sloppy, and some bacteria DNA is excised
along with it. When the resulting virus infects another cell, it will pass that bacterial DNA
into the cell, along with its own DNA. If the infected cell survives (it can happen; there are
bacterial defenses against viral infection), it will contain a new piece of bacterial DNA, which
can undergo recombination and possibly cause gene conversion. Because the viral DNA
integrates into a specific location, when it excises, the bacterial DNA removed with it will be
the same in all cases. Therefore, the DNA transferred to the second cell will be the same
segment of the bacterial chromosome. This is why this process is called 'specialized'
transduction.
Bacterial Recombination: Summary
Bacteria can pick up loose DNA in their environment through the process of
transformation. The newly acquired DNA is rendered single stranded, and can recombine
with the host chromosome.
• Bacteria can exchange DNA through the process of conjugation. The F factor confers
the ability to initiate conjugation. If the F factor alone is transferred, no
recombination will occur. Under certain circumstances, chromosomal DNA can be
transferred to the recipient cell. In these cases, recombination will occur.
• Bacteria can receive bacterial DNA from viruses through the process of transduction.
Bacterial viruses can accidentally pick up pieces of bacterial DNA. When they
subsequently infect a cell, they transfer the pice of bacterial DNA, which can undergo
recombination with the host bacterial chromosome.
• The result of recombination in the above cases may be gene conversion, in which a
mutant allele becomes wild-type or vice versa.
• Conjugation involving Hfr bacteria can be used to map genes along the bacterial
chromosome. This done by determining in what order genes are transferred during
conjugation, waht the time difference is between the transfer of genes.
Bacteria do not reproduce sexually but can acquire new DNA through transformation,
transduction or conjugation.These natural processes have been modified so that DNA can be
deliberately incorporated into host microbes- even genes that would normally never be
transferred this way.
 GENETIC ENGINEERING - PLASMIDS, EPISOMES

Genetic Engineering

Genetic engineering is a laboratory technique used by scientists to change the DNA

of living organisms.

DNA is the blueprint for the individuality of an organism. The organism relies upon
the information stored in its DNA for the management of every biochemical process. The
life, growth and unique features of the organism depend on its DNA. The segments of DNA
which have been associated with specific features or functions of an organism are called
genes.

Molecular biologists have discovered many enzymes which change the structure of
DNA in living organisms. Some of these enzymes can cut and join strands of DNA. Using
such enzymes, scientists learned to cut specific genes from DNA and to build customized
DNA using these genes. They also learned about vectors, strands of DNA such as viruses,
which can infect a cell and insert themselves into its DNA.

With this knowledge, scientists started to build vectors which incorporated genes of
their choosing and used the new vectors to insert these genes into the DNA of living
organisms. Genetic engineers believe they can improve the foods we eat by doing this. For
example, tomatoes are sensitive to frost. This shortens their growing season. Fish, on the
other hand, survive in very cold water. Scientists identified a particular gene which enables
a flounder to resist cold and used the technology of genetic engineering to insert this 'anti-
freeze' gene into a tomato. This makes it possible to extend the growing season of the
tomato.

Plasmids (Video)
A plasmid is an extra chromosomal DNA molecule separate from the chromosomal
DNA which is capable of replicating independently from the chromosomal DNA. In many
cases, it is circular and double-stranded. Plasmids usually occur naturally in bacteria, but
are sometimes found in eukaryotic organisms (e.g., the 2-micrometre-ring in
Saccharomyces cerevisiae).
Plasmid size varies from 1 to over 1,000 kilobase pairs (kbp). The number of
identical plasmids within a single cell can range anywhere from one to even thousands
under some circumstances. The number of plasmids in a cell generally remains constant
from generation to generation.
Properties of Plasmids
• Circular DNA elements, always double-stranded DNA, Supercoiled
• Can occur in as few as 1 copy per cell (single copy plasmids) to as many as several dozen
(multicopy plasmids).  
• Variable sizes; small plasmids about 0.1% size of host chromosome, large plasmids can
be as much as 10% the size of host chromosome. Smaller plasmids have few genes
(30 or less). Size ranges from 1000 bp (1 kbp) to 1000 kbp.
• Ubiquitous; almost all cells isolated in nature carry plasmids, often more than one kind.
(In E. coli alone, more than 300 different plasmids isolated.)
• Have a replicon (origin for DNA replication), number of copies per cell regulated. Large
plasmids typically only 1-5 copies/cell (stringent control); small plasmids ~10-50
copies/cell (relaxed control)
• Many plasmids are incompatible; if one is present, cell cannot support another plasmid of
same compatibility group.
• Not essential to cell under all circumstances; can be "cured" by agents that impair DNA
replication ----> cured cell lacking plasmid. Can be spontaneously lost over time
unless some selection makes plasmid valuable to cell.
• Extend range of environments in which a cell can live (e.g., by degrading antibiotics, or
providing enzymes for digestion of novel catabolites).
Examples of Plasmid genes
• Antibiotic resistance genes (enzymes that modify or degrade antibiotics) -- plasmids with
these genes are called R factors
• Heavy metal resistance (enzymes that detoxify metals by redox reactions)
• Growth on unusual substrates (enzymes for hydrocarbon degradation, etc.)
• Restriction/modification enzymes (protect DNA, degrade unprotected DNA)
• Bacteriocins (proteins toxic to other bacteria lacking the same plasmid)
• Toxins (proteins toxic to other organisms; e.g. humans) -- called virulence plasmids.
Some Examples:
1. Staph aureus virulence factors: coagulase, hemolysin, enterotoxin, others
2. pathogenic E. coli strains: hemolysin, enterotoxin
Proteins that mediate plasmid transfer to uninfected strains
There are two categories of plasmids. Stringent plasmids replicate only when the
chromosome replicates. This is good if you are working with a protein that is lethal to the
cell. Relaxed plasmids replicate on their own. This gives you a higher ratio of plasmids to
chromosome. Some of the traits coded by plasmids include:

Classification of Plasmids
1. Transfer properties
a. Conjugative plasmids - Conjugative plasmids are those that mediated conjugation.
These plasmids are usually large and have all the genes necessary for autonomous
replication and for transfer of DNA to a recipient (e.g. genes for sex pilus).
b. Nonconjugative plasmids - Nonconjugative plasmids are those that cannot mediate
conjugation. They are usually smaller than conjugative plasmids and they lack one or more
of the genes needed for transfer of DNA. A nonconjugative plasmid can be transferred by
conjugation if the cell also harbors a conjugative plasmid.
2. Phenotypic effects
a. Fertility plasmid (F factor)
b. Bacteriocinogenic plasmids - These plasmids have genes which code for substances
that kill other bacteria. These substances are called bacteriocins or colicins.
c. Resistance plasmids 7 factors) - These plasmids carry antibiotic resistance genes.
i) Origin - The origin of the R factors is not known. It is likely that they evolved for other
purposes and the advent of the antibiotic age provided a selective advantage for their wide-
spread dissemination.
ii) Structure - R plasmids are conjugative plasmids in which the genes for replication and
transfer are located on one part of the R factor and the resistance genes are located on
another part as illustrated in Figure.
RTF (Resistance Transfer Factor) - carries the transfer genes.
R determinant - carries the resistance genes. The resistance genes are often parts of
transposons.
Mode of action of resistance genes
a) Modification (detoxification) of antibiotic - e.g. β-lactamase
b) Alteration of target site - e.g. Streptomycin resistance
c) Alteration of uptake - Tetracycline resistance
d) Replacement of sensitive pathway - e.g. new folic acid pathway for resistance to sulfa
drugs.
Plasmids are easy to manipulate and isolate using bacteria. They can be integrated
into mammalian genomes, thereby conferring to mammalian cells whatever genetic
functionality they carry. Thus, this gives you the ability to introduce genes into a given
organism by using bacteria to amplify the hybrid genes that are created in vitro. This tiny
but mighty plasmid molecule is the basis of recombinant DNA technology.
Episome

Episome is a unit of genetic material composed of a series of genes that sometimes

has an independent existence in a host cell and at other times is integrated into a
chromosome of the cell, replicating itself along with the chromosome. Episomes have been
studied in bacteria. One group of episomes are actually viruses that infect bacteria. As
autonomous units they destroy host cells, and as segments integrated into a chromosome
they multiply in cell division and are transferred to daughter cells. Episomes called sex
factors determine whether chromosome material will be transferred from one bacterium to
another. Other episomes carry genes that make bacteria resistant to the inhibitory action of
antibiotics.

Transposons

Are sequences of DNA that can move or transpose themselves to new positions within
the genome of a single cell. The mechanism of transposition can be either "copy and paste" or
"cut and paste". Transposition can create phenotypically significant mutations and alter the cell's
genome size. Barbara McClintock's discovery of these jumping genes early in her career earned
her a Nobel prize in 1983. Transposons make up a large fraction of the C-value of eukaryotic
cells. Transposons are often considered "junk DNA". In Oxytricha, which has a unique genetic
system, they play a critical role its development. Transposons are very useful to researchers as
a means to alter DNA inside a living organism

 
 GENETICALLY MODIFIED ORGANISM
Genetic engineering - was made possible through a series of scientific advances
including the discovery of DNA and the creation of the first recombinant bacteria in 1973,
i.e., E .coli expressing a salmonella gene. This led to concerns in the scientific community
about potential risks from genetic engineering.
A genetically modified organism (GMO) or genetically engineered organism (GEO) is
an organism (plant or animal) whose genetic material has been altered using genetic
engineering techniques. These modifications are generally used to benefit medicine or food
production. There is controversy about these techniques, as there are fears of tampering
with an organism's evolution, and the long-term irreversible effects that could come from
that tampering.
The general principle of producing a GMO is to add a lot of genetic material into an
organism's genome to generate new traits. These techniques, generally known as
recombinant DNA technology, use DNA molecules from different sources, which are
combined into one molecule to create a new set of genes. This DNA is then transferred into
an organism, giving it modified or novel genes. Transgenic organisms, a subset of GMOs,
are organisms which have inserted DNA that originated in a different species.
Genetically modified bacteria were the first organisms to be modified in the
laboratory, due to their simple genetics. These organisms are now used for several
purposes, and are particularly important in producing large amounts of pure human proteins
for use in medicine.The first example of this occurred in 1978 when Herbert Boyer working
at a University of California laboratory took a version of the human insulin gene and
inserted into the bacterium Escherichia coli to produce synthetic "human" insulin.The drug
industry has made good use of this discovery in its quest to cure diabetes. Similar bacteria
have been used to produce clotting factors to treat haemophilia, and human growth
hormone to treat various forms of dwarfism. These recombinant proteins are safer than the
products they replaced, since the older products were purified from cadavers and could
transmit diseases. Indeed the human-derived proteins caused many cases of AIDS and
hepatitis C in haemophilliacs and Creutzfeldt-Jakob disease from human growth hormone.
For instance, the bacteria which cause tooth decay are called Streptococcus mutans. These
bacteria consume leftover sugars in the mouth, producing lactic acid that corrodes tooth
enamel and ultimately causes cavities. Scientists have recently modified Streptococcus
mutans to produce no lactic acid. These transgenic bacteria, if properly colonized in a
person's mouth, could reduce the formation of cavities. Transgenic microbes have also been
used in recent research to kill or hinder tumors, and to fight Crohn's disease. Genetically
modified bacteria are also used in some soils to facilitate crop growth, and can also produce
chemicals which are toxic to crop pests.

Moving Genes between Species—how to do……

• The process by which scientists introduce new genetic material into a
microorganism is called molecular or gene cloning or genetic engineering.
• It involves the isolation of DNA from a source other than the microorganism itself.
Source organisms span the world of living things, from microbes to plants to
animals, including humans. Scientists obtain source DNA in several different ways:
by disrupting cells of the target microbe (or plant or animal) and fragmenting
it into small pieces, by synthesizing it from an RNA template using an
enzyme called reverse transcriptase, or by knowing the specific gene
sequence and synthesizing it directly in the laboratory.
• Once obtained, the pieces of DNA are inserted into a small genetic component that
has the ability to make copies of itself (replicate) independently from the microbial
genome. This self-replicating unit is called a cloning vector. Although these genetic
elements exist naturally in the form of plasmids and bacterial viruses, many of
the ones used today have been altered to improve their properties for transferring
genes. Restriction enzymes, which nick the donor DNA and the cloning vector at
specific sites, and DNA ligase, which attaches the donor DNA to the cloning vector,
allow the source genes of interest to be inserted into the cloning vector without
disrupting its ability to replicate.
• The next step in the process is the introduction of the cloning vector with its
segment of new DNA into a living cell. Bacteria have the ability to transport DNA
into their cells in a process called transformation, and this ability is commonly
exploited to achieve this goal. Getting the DNA into the cell, however, is only the
beginning. No transformation is 100 percent efficient, and so the bacteria that
receive the gene(s) of interest must be separated from those that did not. One of
the best studied and most commonly used cloning vectors, pBR322, is especially
useful for this purpose, as it contains several genes for antibiotic resistance.
Hence, any cell transformed with DNA containing pBR322 will be antibiotic
resistant, and thus can be isolated from similar cells that have not be so
transformed by merely growing them in the presence of the appropriate drugs. All
that remains is to identify bacteria that are producing the product of the desired
gene(s), and cloning is a success.
 • The introduction of human genes into bacteria has several complicating wrinkles
that make cloning them even more challenging. For example, a bacterial gene
codes for a protein from start to finish in one long string of nucleotides, whereas
human cells have stretches of noncoding nucleotides called introns within their
genes. Bacteria do not have the same ability as human cells to remove these
introns when producing proteins from the gene, and if the introns are not
removed, the intended protein cannot be produced. This, along with other
complications, has been overcome using many of the tools of genetic engineering.
Uses of GMOs
Examples of GMOs are highly diverse, and include transgenic (genetically modified
by recombinant DNA methods) animals such as mice, fish, transgenic plants, or various
microbes, such as fungi and bacteria. The generation and use of GMOs has many reasons,
chief among them are their use in research that addresses fundamental or applied questions
in biology or medicine, for the production of pharmaceuticals and industrial enzymes, and
for direct, and often controversial, applications aimed at improving human health (e.g.,
gene therapy) or agriculture (e.g., golden rice). The term "genetically modified organism"
does not always imply, but can include, targeted insertions of genes from one into another
species. For example, a gene from a jellyfish, encoding a fluorescent protein called GFP, can
be physically linked and thus co-expressed with mammalian genes to identify the location of
the protein encoded by the GFP-tagged gene in the mammalian cell. These and other
methods are useful and indispensable tools for biologists in many areas of research,
including those that study the mechanisms of human and other diseases or fundamental
biological processes in eukaryotic or prokaryotic cells.

Transgenic microbes
Bacteria were the first organisms to be modified in the laboratory, due to their
simple genetics. These organisms are now used in a variety of tasks, and are particularly
important in producing large amounts of pure human proteins for use in medicine.

Bacteria-synthesized transgenic products

• Insulin
• Interferon
• Hepatitis B vaccine
• Tissue plasminogen activator
• Human growth hormone
• Ice-minus bacteria
 Did you know that this is a is a nickname given to a variant of the common
bacterium Pseudomonas syringae (P. syringae). This strain of P. syringae lacks the
ability to produce a certain surface protein, usually found on wild-type P. syringae.
The "ice-plus" protein (In a protein, "Ice nucleation-active" protein) found on the
outer bacterial cell wall acts as the nucleating centers for ice crystals. This facilitates
ice formation, hence the designation "ice-plus." The ice-minus variant of P. syringae
is a mutant, lacking the gene responsible for ice-nucleating surface protein
production. This lack of surface protein provides a less favorable environment for ice
formation. Both strains of P. syringae occur naturally, but recombinant DNA
technology has allowed for the synthetic removal or alteration of specific genes,
enabling the creation of the ice-minus strain. Modifying P. syringae may have
unexpected consequences for climate. A study has shown that its ice nucleating
proteins may play an important part in causing ice crystals to form in clouds. If
humans increase the frequency of bacteria lacking these proteins then it may affect
rainfall

Commercial Applications
• Transgenic microbes have many commercial and practical applications, including
the production of mammalian products. A company called Genentech was among
the earliest and most successful commercial enterprises to use genetically
engineered bacteria to produce human proteins. Their first product was human
insulin produced by genetically engineered Escherichia coli. A variety of other
human hormones, blood proteins, and immune modulators are now produced in a
similar fashion, in addition to vaccines for such infectious agents as hepatitis B
virus and measles.
• Another promising application of genetically engineered microbes is in
environmental cleanup, or biomediation. Scientists have discovered many
naturally occurring genes that code for enzymes that degrade toxic wastes and
wastewater pollutants in bacteria. Examples include genes for degrading
chlorinated pesticides, chlorobenzenes, naphthalene, toluene, anilines, and various
hydrocarbons. Researchers are using molecular cloning to introduce these genes
from several different microbes into a single microbe, creating "super microbes"
with the ability to degrade multiple contaminants.
• Ananda Chakrabarty created one of the first microbes of this nature in the early
1970s. He introduced genes from several different bacteria into a strain of
Burkholderia cepacia, giving it the ability to degrade toxic compounds found in
 petroleum. This microbe offered a potential alternative to skimming and absorbing
spilled oil. Chakrabarty's genetically modified bacterium has never been used,
however, due to public concerns about the release of genetically engineered
microbes into the environment. The microbe did, on the other hand, play an
important role in establishing the biotechnology industry. The U.S. Patent Office
granted Chakrabarty the first patent ever for the construction and use of a
genetically engineered bacterium. This established a precedent allowing
biotechnology companies to protect their "inventions" in the same way chemical
and pharmaceutical companies have done in the past.

In addition to bacteria being used for producing proteins, genetically modified viruses
allow gene therapy. Gene therapy is a relatively new idea in medicine. A virus reproduces
by injecting its own genetic material into an existing cell. That cell then follows the
instructions in this genetic material and produces more viruses. In medicine this process is
adapted to deliver a gene that could cure disease into human cells. Although gene therapy
is still relatively new, it has had some successes. It has been used to treat genetic disorders
such as severe combined immunodeficiency, and treatments are being developed for a
range of other incurable diseases, such as cystic fibrosis, sickle cell anemia, and muscular
dystrophy.
Genetically modified bacteria are also used in agriculture to facilitate crop growth,
and can also produce chemicals which are toxic to crop pests.

Transgenic animals
Transgenic animals are used as experimental models to perform phenotypic tests
with genes whose function is unknown or to generate animals that are susceptible to certain
compounds or stresses for testing in biomedical research. Other applications include the
production of human hormones, such as insulin. Frequently used in genetic research are
transgenic fruit flies (Drosophila melanogaster) as genetic models to study the effects of
genetic changes on development. Transgenic mice are often used to study cellular and
tissue-specific responses to disease.

Transgenic plants
Transgenic plants have been developed for various purposes. Most of transgenic
plants were created for research purposes and were not intended for eventual
commercialization. From these few which have reached the market the most common
transgenic traits include 1) resistance to pests or herbicides, 2) improved product shelflife.
In the near future crops with improved nutritional value and with resitance to harsh
environmental conditions might reach the marketplace. Since the first commercial
cultivation of GM plants in 1996, GM plants tolerant to the herbicides glufosinate or
glyphosate, and producing the Bt toxin, an insecticide, have dominated the agriculutral seed
market for corn and other crops (soybean, cotton). Recently, a new generation of GM plants
promising benefits for consumers and industry purposes is entering the market. Whenever
GM plants are grown on open fields without containment there are risks that the
modification will escape into the general environment. Most countries require biosafety
studies prior to the approval of a new GM plant release, usually followed by a monitoring
programme to detect environmental impacts. Especially in Europe, the coexistence of GM
plants with conventional and organic crops has raised many concerns. Since there is
separate legislation for GM crops and a high demand from consumers for the freedom of
choice between GM and non-GM foods, measures are required to separate foods and feed
produced from GMO plants from conventional and organic foods. European research
programmes such as Co-Extra, Transcontainer and SIGMEA are investigating appropriate
tools and rules.
Controversy over GMOs
The use of GMOs has sparked significant controversy in many areas. Some groups or
individuals see the generation and use of GMO as intolerable meddling with biological states
or processes that have naturally evolved over long periods of time (although many crops
and animals have been modified by humans via unnatural selection over the last several
thousand years), while others are concerned about the limitations of modern science to fully
comprehend all of the potential negative ramifications of genetic manipulation.
While some groups advocate the complete prohibition of GMOs, others call for
mandatory labeling of genetically modified food or other products. Other controversies
include the definition of patent and property pertaining to products of genetic engineering
and the possibility of unforeseen local and global effects as a result of transgenic organisms
proliferating. The basic ethical issues involved in genetic research are discussed in the
article on genetic engineering.
 SOIL MICROBIOLOGY: MICROBIAL GROUPS IN SOIL

The field of soil microbiology was explored during the very last part of 19th century.
The establishment of the principal roles that microorganisms play in the biologically
important cycles of matter on earth: the cycles of nitrogen, sulphur and carbon was largely
the work of two men, S. Winogradsky (1856-1953) and M.W. Beijerinck (1851–1931). S.
Winogradsky, Russian and regarded by many as the founder of soil microbiology,
discovered nitryfiying bacteria (1890-91); described the microbial oxidation of H2S and
sulphur (1887); developed the contributed to the studies of reduction of nitrate and
symbiotic nitrogen fixation; and, originated the nutritional classification of soil
microorganisms into autochtonous (humus utilizers) and zymogenous (opportunistic)
groups.
Almost equally important was the work of M.W. Beijerinck, a Hollander, who isolated
the agents of symbiotic (1888) and non-symbiotic aerobic (1901) nitrogen fixation.
However, the greatest contribution of Beijerinck was a new and profoundly important
technique: enrichment culture technique: to isolate and study various physiological types of
various microorganisms from natural samples through the use of specific culture media and
incubation conditions.

Bacteria- more dominant group of microorganisms in the soil and equal to one half
of the microbial biomass in soil. Majority are Heterotrophs. (Common soil bacteria -
Arthrobacter, Bacillus, Clostridium, Micrococcus).
ROD SHAPED BACTERIA

SPHERICAL BACTERIA

SPIRILLUM
 Actinomycetes - intermediate group between bacteria and fungi. Numerous and
widely distributed in soil. Abundance is next to bacteria. 104 - 108/g soil. 70% of soil
actinomycetes are Streptomyces. Many of them are known to produce antibiotics.
Population increases with depth of soil.
 Fungi: More numerous in surface layers of well-aerated and cultivated soils-
dominant in acid soils. Common genera in soil are Aspergillus, Mucor, Penicillium
Trichoderma, Alternaria, Rhizopus.
Algae – found in most of the soils in number ranges from 100 to 10,000 per g.
 Protozoa: Unicellular – population ranges from 10,000 to 100,000 per g of soil.
Most of the soil forms are flagellates, amoebae or ciliates. Derive their nutrition by
devouring soil bacteria. Abundant in upper larger of the soil. They are regulating the
biological equilibrium in soil.

Importance
• Involved in nutrient transformation process
• Decomposition of resistant components of plant and animal tissue
• Role in microbial antagonism
• Participate in humus formation
• Predator of nematodes
• Surface blooming reduces erosion losses
• Improve soil structure
• Involved soil structure
• Maintenance of biological equilibrium
Factors influencing activities of soil microorganisms
Soil microorganisms are influenced by various factors. Chief factors are,
• fertility level
• Soil moisture
• Soil air
• soil temperature
• Organic matter
• H ion concentration
• Cultural factors
 MICROBIAL TRANSFORMATIONS OF CARBON
The term soil generally refers to the loose material of the earth surface and is the region that
supports the plant life. It consists of five major components such as mineral matter, water, air,
organic matter and living organisms. The proportion of these components varies with soil type
and other soil conditions. To maintain the level of these components it is essential that they
undergo a regular process of recycling. This process of recycling through various
transformations is brought about by different microorganism.
Carbon cycle (Video)

Plant carbon Animal carbon

C
Soil organic matter

B
A
 

Microbial cell and decayed

D residuesE

Carbon dioxide

A- PS

B- Respiration / plant

C- Respiration / Animals

D- Autotrophs

E- Respiration
The / Microbial
most important element inmineralization
the biological realm and substance that serve as the
cornerstone of the cell structure is carbon. It constituents about 40-50% of all living organisms,
yet the ultimate source is the CO2 that exist in a perennially short supply, only 0.03% of the
earth’s atmosphere, which undergo a cyclic change from an oxidized to reduced state.
 Carbon (CO2) is constantly (reduced into organic carbon compounds) being fixed into
organic form by photosynthetic organisms (photosynthesis). Once bound, the carbon becomes
unavailable for use in generation of new plant life. It is thus essential for the carbonaceous
materials to be decomposed and returned to the atmosphere. It is estimated that 1.3x1014 kg
CO2 is fixed annually in the biosphere. To the lesser extent CO2 is also fixed through the
agency of photosynthetic bacteria and other chemolithotrophs with the convertion of so much of
the plant available carbon to organic form each year and the limited supply in the air, it is
apparent that the major plant nutrient element would become exhausted in the absence of
microbial transformation.
The carbon cycle revolves about CO2 and its fixation and regeneration. The green plants
utilize CO2 as their sole carbon source, and the carbonaceous matter synthesized serves to
supply carbon to other heterotrophic organisms and animals. Upon the death of plants and
animals, microbes assume a dominant role in carbon cycle. The dead tissues are degraded
and transformed into microbial cells and humus or soil organic fraction. Further decomposition
of these materials leads to the production of CO2 and once again it is recycled.
Organic matter decomposition (Aerobic decay)
Soil organic matter
The organic matter subjected to microbial decay in soil comes from several sources like
plant remains, forest litter, incorporation of plant and animal tissues and excretory products.
The chemistry of organic matter is clearly very complex, and investigations of the
transformations and the responsible organisms have therefore been extremely interesting. The
organic constituents of the plants are commonly divided into six categories.
a) Cellulose - Most abundant 15-60% of the dry weight
b) Hemicellulose - 10-30% of the plant dry weight
c) Lignin - 5 – 30 % of the plant dry weight
d) Water soluble fraction - 5-30%, included simple sugar, a. acids,
e) Ether and alcohol soluble constituents, a fraction containing fat, oils, waxes, resins and a
number of pigments
f) Proteins.
As the plant ages, the content of water soluble constituents, proteins and minerals
decreases and the % of abundance of cellulose, hemicellulose and lignin rises.
Soil organic matter comprises residues of plant and animals and these compounds
occur in soil in close combination with inorganic substances. Animals and plant residues are
made up of complex carbohydrates, simple sugars, starch, cellulose, hemicellulose, pectins,
gums, mucilage, proteins fats, oils, waxes, resins, alcohols, aldehydes , ketones, organic acids,
lignin, phenols, tannins, hydrocarbons, alkaloids, pigments etc.
• The soil microorganism play important role in the decomposition of soil organic matter.
• Bacteria are the dominant group – mostly heterotrophic organisms (use energy from
organic sources such as sugars, starch, cellulose and protein) – are involved.
Autotrophic organism which occupy a small portion of the biomass in soil (and use
inorganic sources such as Fe and S) are not directly involved in organic matter
decomposition.
• Actinomycetes grow on complex substances such as keratin, chitin and other complex
polysaccharides and play active role in humus formation.
• Soil fungi are mostly heterotrophos and use organic residues easily
• Soil algae contribute a small amount of organic matter through their biomass, but they
do not have any active role in organic matter decomposition.
Organic matter decomposition serves two important functions
a) Provide energy for growth
b) Supply carbon for the formation of new cell materials
Hence only heterotrophs are actively involved in the process of decomposition. The
relationship between organic matter and plant growth may be direct or indirect.
• Organic matter is a natural substrate for saprophytic micro organism and provides
nutrition to plants indirectly through the activity of soil microorganisms
• It is essential for the formation of soil aggregates and hence soil structure which
ultimately determines the soil aeration and rooting habit of plants
• Organic matter helps in the conservation of soil nutrients by preventing erosion and
surface run off of nutrients.
Carbon assimilation
The process of converting substrate to protoplasmic carbon is known as assimilation.
Under aerobic conditions 20-40% of the substrate carbon is assimilated, the remainder is
released as CO2. Fungi are more efficient, in their metabolism, since they convert carbon into
cell carbon as filaments and release less of CO2. 30-40% of which is used to form new
mycelium during the decomposition. Compared to fungi, bacteira are less efficient. Aerobic
bacteria are less efficient than anerobic bacteria.
C. Mineralization
- Conversion of organic Carbon substance to inorganic form of carbon.

Immobilization
Assimilation of nutrients and is the mechanism by which micro organism reduce the
quantity of plant available nutrient in soil. Mineralization is considered well than immobilization.
During the decomposition of organic matter three separate simultaneous processes can
be distinguished. The important changes during decomposition are:
1. Plant and animal tissues constituents disappear under the influence of enzymes
2. Synthesis of new microbial cells so that proteins, polysaccharides and nucleic acids
typical of bacteria and fungi appear.
3. Third, certain end products of the breakdown are excreted into surroundings there to
accumulate or to be further metabolized.
Importance of organic matter decomposition
1. Important function is the breakdown of organic matter by which CO2 available for
photosynthesis is replenished
 2. Any compound that is synthesized biologically is subject to destruction by soil
inhabitants, otherwise the compounds would have accumulated in vast amounts on the
earth’s surface
3. Since, organic matter degradation is a property of all heterotrophs, it is commonly used
to indicate the level of microbial activity.
Methods to evaluate the decomposition rate
a) Measurement of CO2 evolution or O2 uptake
b) Determination of decrease in organic matter either chemically or by weight loss
c) Observations on disappearance of specific constituents such as cellulose, hemicellulose
or lignin.
Changes during organic matter decomposition
As a result of development of mixed flora on chemically complex natural products, some
components quickly disappear while others are less susceptible to microbial enzymes and
persist. The water soluble fraction contains the least resistant plant components and is thus the
first to be metabolized. Cellulose and hemicellulose on the other hand disappear not as quickly
as water soluble substances, but their persistence usually is not too great. The lignins are
highly resistant and consequently become relatively more abundant in the residual, decaying
organic matter.
• At aerobic conditions when carbonaceous substrates are incorporated into soil,
immediate drop in O2 and an increase in CO2 content of soil air occurs.
• Change in (O) (H) oxidation reduction potential (En) – it is shifted to a more reduced
condition (fall in oxidation reduction potential).
The end products of decomposition are - CO2, H2O, NO3, SO4, CH4, NH4 and H2S depending
on the availability of air.
Factors influencing the organic matter decomposition
- Organic matter level of the soil
- Cultivation
- Temperature
- Moisture
- pH
- Depth
- Aeration
- Nature and abundance of micro organic involved.
- The extent of availability of C, N, P and K presence of inhibitory substance.
C: N ratio
• Nitrogen is a key nutrient substance for microbial growth
• If N content of the substrate is high it is readily utilized and decomposition is faster
• If N is poor decomposition is slower, needs additional N
• Protein rich substrates are readily decomposed
• Low N or wide C:N ratio results slow decay
• Optimum level of C: ratio for maximum decomposition is 20-25(1.4-1.7% N)
• Less than this range, more microbial cells, faster mineralization and it likely exceeds
immobilization
• Wider the ratio, lesser microbial cells, slower the immobilization and mineralization
increases gradually, resulting in accumulation of Ammonia and Nitrates
• Microbes scavenge the soil solution to obtain enough N
• At optimum level, there must be an equilibrium between Mineralization and
Immobilization
• Soil N level constrains the maintenance of C:N (organic carbon /soil o.m)
• To make sound soil management
• Arable surface -10:1
o Sub soil -lower
Anaerobic decay / decomposition
The main products of aerobic carbon mineralization are CO2, water, cells and humus
components. In the absence of O2 organic carbon is incompletely metabolized, intermediary
substances accumulate, and abundant quantities of CH4 and smaller amounts of H2 are
evolved. Energy yield during anaerobic fermentation is low, resulting in fewer microbial cells per
unit of organic carbon degraded. Consequently, organic matter breakdown is consistently
slower under total anaerobiosis than in environments containing adequate O2. The rate in water
logged soils is intermediate between the two extremes.
When a soil is water logged or flooded there is a shift from aerobic to anaerobic
transformation. Formation and accumulation of organic acids viz., acetic, formic, butyric, lactic
and succinic acids appear too, these are frequently detrimental to root development. Organic
acids accumulate because of the fermentative character of the microflora of wet soils. The an
aerobic carbon transformation are thus characterized by the formation of organic acids,
alcohols, CH4 and CO2 as major end products.
Under anaerobic conds, decomposition of organic residues takes place by the activity of
both mesophilic, thermophilic microorganism resulting in the production of CO2, H, ethyl alcohol,
and organic acids. Among mesoophilis, bacteria are more active than fungi or actinomycetes in
cellulolytic activity. They belong to genus Clostridium which are numerous in manure pits but
rarely encountered in cultivated arable soils. In compost pits both meso and thermopholic
(bacterial and actinomycetes) are important in the breakdown of cellulose substances.
The primary microbial colonisers initially break down the complex CH2O and proteins
into organic acids and alcohols. At a later stage, the methane bacteria which are strict
anaerobes begin to act upon the secondary substrate chiefly lactic and butyric acids and
ferment them into CH4 and CO2.
Humus
• A dark coloured and fairly stable soil organic matter with known and unknown physical
and chemical properties
• It is an integral part of the organic matter complex in soil
• Humus can be defined as lingo protein complex containing approximately
45 % - lignin compounds
35% - amino acids
11% - carbohydrates
4% - cellulose
7% - Hemicellulose
3% - fats, wax, resins
6% - other miscellaneous substances, including plant growth substances and inhibitors.
• Age and composition of the humus are dependent on its origin and environment.
• Bacterial and algal protoplasm contribute a good deal to the nutritive value of humus
• Soil micro organism take part in humus formation. Some fungi such as Penicillium,
Aspergillus and actinomycetes produce dark humus like substances which serve as
structural units for the synthesis of humic substances.
Benefits of humic substances
• Improved seed germination, root growth, uptake of minerals by plants and other
physiological effects on plant growth
• Increases the enzyme activities involved in plant metabolism. Since humic acid serves
as hydrogen acceptors.
• Increases the cytochrome oxidase activity in root systems results in growth stimulatory
effect (on roots)
• Chelating effect – on trace elements Fe uptake by roots
• Vigour and yield of plants enhanced
 • Humic acid known to influence the grown and proliferation of micro organism
• Aspergillus niger, Peni, Bacillus sp., Azotobacter are enhanced
The organic fraction of soil, often termed humus. It is a product of synthetic and
decomposing activities of the microflora. Since it contains the organic C and N needed for
microbial development, it is the dominant food reservoir. Because humus is both a product of
microbial metabolism and an important food source, the organic fraction is of special interest.
Humus formation
• Once the plant or animal remains fall on or are incorporated into the soil, they are
subjected to decomposition
• From the original residues, a variety of products are formed
• As the original materials and the initial products undergo further decomposition, they are
converted to brown or black organic complexes
• At this stage any trace of the original remains no longer remains
• The native organic fraction originates from two sources: the original plant debris entering
the soil and the micro organism with in the soil body. The micro organism in soil body,
work upon the former and synthesize microbial protoplasm and new compounds that
become part of the organic fraction.
• Humus exist in a dynamic state
• Chemistry of humus is complex
• It has been pointed out that the organic fraction is derived from
a) Plant constituents that are modified by the microflora.
b) Constituents of microbial cells and products of microbial metabolism are
relatively resistant to decay and therefore persist for sometime after death of
organism.
Interms of specific elements
The organic fraction contains compounds of C, H, O, N, P, S and small amounts of other
elements. Only a small portion of the total is soluble in water, but much can be brought into
solution by alkali.
Interms of type of compounds
Humus contains a number of polymerized substances, aromatic, molecules,
polysaccharides, ascorbic acids, polymers of uronic acids and P containing compounds.
No definite composition can be assigned. It should be considered as a portion of the soil
that is composed of a heterogenous group of substances, most having an unknown parentage
and an unknown chemical structure.
 Lignin and lignin derived molecules have long been considered to be of significance in
the formation of humus.
It is possible either that simple aromatics released in the microbial attack on lignin
polymerize to yield constituents of the soil organic fraction or those partially altered lignins itself
give rise to humus constituents. The monomeric portions of humus are similar to the
constituents of lignin.
Degradation processes
(1) Cellulose is a CH2O composed of glucose units bound by β-linkage at carbon 1 and 4 of the
sugar molecule. The cellulose concentration of higher plants is never fixed and the
concentration. It is a polymer of glucose and is might abundant organic material in nature
changes with age and type of plants. Woody materials have more cellulose and succulent
tissues had poor, but the concentration increases as the plant matures. Cellulose breakdown in
soil is influenced by several environmental factors.
Aerobic organism converts cellulose to 2 major products: CO2 + cell substance, but
certain group releases small amount of organic acids. It is however resistant to microbial
decomposition. When cellulose is associated with pentosans (xylan, mannans) it undergoes
rapid decomposition. When associated with lignin, the decomposition rate is very low.
Degradation is by the enzymes that converts cellulose into glucose. (Exoenzymes)
Exoglucanase
Endoglucanase
β - glucosidase (cellulse complex)

Exo glucanase
Native cellulose Amorphos cellulose + cellobiose
Endoglucanase Endogluconase

β-glucosidase
D- Glucose Cellobiose
(cellobiase)
Most cellulolytic bacteria do not excrete significant amounts of cellulase but fungi are
found to excrete these enzymes. The soluble sugars released by enzymatic hydrolysis are later
utilized by the same or other micro organism for biosynthetic purpose.
(2) Hemicellulose
 It is a polymer of simple sugars such as pentose, hexose and uronic acids. They may
be either homo or hetero polymers. When they are added to soil, degradation takes place at
faster rate in initial stages. The hemicelluloses such as mannans are decomposed rapidly while
galactons (polymer of galactose) are decomposed slowly. Many soil micro organisms utilize
hemicellulose in both aerobic as well as anaerobic conditions. The microbial degradation occurs
through the agency of extracellular enzymes called hemicellulases.
(3) Lignin
Lignin is the third most abundant costituent of plants. It consists of heterocyclic aromatic
organic molecules containing C, H and O. The degradation is very slow and rate of
decomposition depends on the presence of other compounds such as cellulose and
hemicellulose acid. Lignin is highly resistant to microbial degradation. Degradation is a
complex process.

• Lignin -- coniferyl ether -- coniferyl alcohol -- coniferyl aldehyde -- vanillin --
 vannillic acid -- protocatechuic acid -- ring cleavage

Genera of microorganisms capable of utilizing different components of organic mater as

reported by several workers: F-Fungi; B-Bacteria; A-Actinomycetes
Nature of substrate in
Genera of microorganisms
organic matter
Cellulose F Alternaria, Aspergillus, Chaetomium, Coprinus, Fomes,
Fusarium, Myrothecium, Penicillum, Polyporus,
Rhizoctonia, Rhizopus, Trametes, Trichoderma,
Trichothecium, Verticillium, Zygorynchus
B Achrombacter, Angiococcus, Bacillus, Cellfalcicula,
Cellulomonas, Cellvibrio, Clostrium, Cytophaga,
Polyangium, Pseudomonas, Sorangium, Sporocytophaga,
Vibrio
A Micromonospora, Nocardia, Streptomyces,
Streptosporangium
Hemicellulose F Alternaria, Fusarium, Trichothecium, Aspergillus,
Rhizopous, Zygorynchus, Chateomium,
Helminthosporium, Penicillium, Coriolus, Fomes,
 Polyporus
B Bacillus, Achromobacter, Pseudomonas, Cytophaga,
Sporocytophaga, Lactobacillus, Vibrio
A Streptomyces
Lignin F Clavaria, Clitocybe, Collybia, Flammula Hypholoma,
Lepiota, Mycena, Pholiota, Arthrobotrys, Cephalosporium,
Humicola
B Pseudomonas, Flavobacterium