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Botany
First Year

MBOTN- 13

MICROBIOLOGY IMMUNOLOGY AND PLANT

PATHOLOGY

SCHOOL OF SCIENCES
TAMIL NADU OPEN UNIVERSITY
577, ANNA SALAI, SAIDAPET, CHENNAI - 15

April, 2022
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Department of Botany
School of Sciences
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 MBOTN - 13: MICROBIOLOGY IMMUNOLOGY AND PLANT
PATHOLOGY

BLOCK I: GENERAL MICROBIOLOGY

Unit 1:History and concepts of Microbiology

Unit 2: History of Microscope

Unit 3: Classification and Ultra Stricture of Bacterial Cell

Unit 4: Bacterial Staining

Unit 5: Bacterial Reproduction

BLOCK II: APPLIED MICROBIOLOGY

Unit 6: Spoilage of Food

Unit 7: Microbes in Fermented Products

Unit 8: Microbes in sewage treatment and Agriculture

Unit 9: Industrial Application of Microbes

BLOCK III: GENERAL VIROLOGY

Unit 10: History of Viruses

Unit 11: Classification and Structure of Virus

Unit 12: Transmission and Isolation of Virus

Unit 13: Life cycle of Virus

Unit 14: Importance of Virus and Special Study

BLOCK IV: IMMUNOLOGY

Unit 15: Introduction to Immunity

Unit 16: Immune System

Unit 17 The Immune Response and Immunoglobulins

Unit 18: Antigen –Antibody Reaction

BLOCK V: PLANT PATHOLOGY

Unit 19: History Scope and Principles of plant infection

Unit 20: Principles of plant infection

Unit 21: Structural and Biochemical Defence Mechanism in Plants

Unit 22: Disease Resistance and incorporation of resistant genes

Unit 23: Methods for incorporation of resistant

BOOKS FOR REFERENCE:

1. Pelczar, Chan and Krieg, 1986, Essentials of Microbiology

2. Dube, H., 1978, A text book of Fungi, Bacteria and Virus. Vikas Publishers.

3. Prescott's Microbiology, by Joanne Willey, Linda Sherwood and Christopher J.

Woolverton9th Edition

4. Brock Biology of Microorganisms, 14th Edition by Michael T. Madigan, John M.

Martinko, Kelly S. Bender, Daniel H. Buckley, David A. Stahl and Thomas Brock

5. Immunology by Thomas J. Kindt, 2002.

6. Cellular and Molecular Immunology by Abul K. Abbas 1991.

7. Agrios, A.G. 2007. Plant Pathology, Elsevier. ISBN: 9780120445653.

8. Singh, R.S. 2018. Introduction to Principles of Plant Pathology, 4 th Edition.

9. Mehrotra, R.S. and Aggarwal, A. 2017. Plant [Link] Hill Publisher.

10. Chaube, H.S. and Singh, R. 2015. Introductory Plant Pathology CBS Publishers,
ISBN: 978-8123926704.

11. Ravi Chandra, N.G. 2013. Fundamentals of Plant Pathology, Phi Learning, ISBN:
812034703X.
 MBOTN - 13 MICROBIOLOGY IMMUNOLOGY AND PLANT PATHOLOGY

SCHEME OF LESSONS

BLOCK I: General Microbiology Page No.

Unit 1: History and concepts of Microbiology 2-11
Unit 2: History of Microscope 12-25
Unit 3: Classification and Ultra Stricture of Bacterial Cell 26-46
Unit 4: Bacterial Staining 47-57
Unit 5: Bacterial Reproduction 58-67

BLOCK II: Applied Microbiology

Unit 6: Spoilage of Food 69-81
Unit 7: Microbes in Fermented Products 82-86
Unit 8: Microbes in sewage treatment and Agriculture 87-106
Unit 9:Industrial Application of Microbes 107-118

BLOCK III: General Virology

Unit 10: History of Viruses 120- 129
Unit 11: Classification and Structure of Virus 130 - 141
Unit 12:Transmission and Isolation of Virus 142 - 156
Unit 13:Life cycle of Virus 157 - 162
Unit 14:Importance of Virus and Special Study 163 - 179

BLOCK IV: Immunology

Unit 15: Introduction to Immunity 181- 196
Unit 16: Immune System 197 - 212
Unit 17: The Immune Responseand Immunoglobulins 213 - 219
Unit 18: Antigen –Antibody Reaction 220 - 228

BLOCK V: Plant Pathology

Unit 19: History Scope and Principles of plant infection 230- 237
Unit 20: Principles of plant infection 238-244
Unit 21: Structural and Biochemical Defence Mechanism in Plants 245-255
Unit 22::Disease Resistance and incorporation of resistant genes 256-261
Unit 23: Methods for incorporation of resistant genes 262-273
 Block I
GENERAL MICROBIOLOGY

Unit 1: History and concepts of Microbiology

Unit 2: History of Microscope

Unit 3: Classification and Ultra Stricture of Bacterial Cell

Unit 4: Bacterial Staining

Unit 5: Bacterial Reproduction

UNIT – 1

HISTORY AND CONCEPTS OF

MICROBIOLOGY
STRUCTURE

Overview

Learning Objectives
1.1 Introduction
1.2 Kosch Pastulate
1.3 Pasteur’s Epic Experiments
1.4 Scope of Microbiology
1.5 Branches of Microbiology
Let us Sum up
Check your Progress
Glossary
Suggested Readings
Answers to check your progress
Model questions

OVERVIEW
Microbes - bacteria, archaea, fungi, algae, protozoa and viruses -
have been around for at least 3,500 million years and were the only life
forms on Earth for most of that time. As the Earth cooled, liquid water formed
and the first microbial life appeared. The conditions on Earth in the beginning
were very hostile so the first microbes probably resembled the archaea, as
they were able to live in the extreme environments such as the high
temperature found on the cooling planet. Microbes affect every aspect of life
on earth. They have an amazing variety of shapes and sizes and can exist in

2
a wide range of habitats from hot springs to the icy wastes of Antarctica and
inside the bodies of animals and plants.

LEARNING OBJECTIVES
To understand the microscopic organisms and its history

1.1 INTRODUCTION

The science of microbiology is the study of microorganisms and their

activities. It is concerned with the form, structure, metabolism, growth,
reproduction and identification of microorganisms. It also includes the study
of their distribution in nature, their relationship to each other and to other
living organisms. For the most part, microbiology deals with microscopic
organisms. Microbiologists are those who specialize to work with the
microorganisms. They have been remarkably successful in exploiting the
useful microorganisms and combating the harmful ones and have also
successfully solved intricate problems of biochemistry and genetics using
microorganisms as tool for their study. Microbiologists may specialize in the
study of different groups of microorganisms. For example, Bacteriology is the
study of bacteria often broadly designated as Microbiology; Mycology is the
study of fungi; Phycology is the study of algae; Protozoology is the study of
protozoa; and Virology is the study of viruses.

Microbiology or study of microorganisms has an interesting past

history. During the thirteenth century Roger Bacon suggested that disease is
induced by invisible living organisms. Similar suggestion was made by
Fracastoro of Verona (1483-1553) and von Plenciz (1762) without any
evidence. But in the mean time in 1658 Kircher designated the disease
inducing living organisms as ‘worms’, which according to him are invisible to
the naked eye. It was Kircher who first recognized the importance of micro-
organisms in disease development. But Antony van Leeuwenhoek (1632-
1723) was the first person to report descriptions of microorganisms in detail.
His discovery brought inspiration to many workers to take interest in the
origin of living things. The concept of origin of animals spontaneously from
the soil, plants, or other unlike animals sponsored by Aristotle (384-322 B.C.)

3
was still accepted in the seventeenth century. In course of time John
Needham (1713-1781), Lazaro Spallanzani (1729-1799), Franz Schulze,
(1815-1873) and Zheodor Schwann (1810-1882), Pouchet (1859) spoke for
and against the theory that living things can originate spontaneously.

Finally Louis Pasteur (1822-1895) through his series of experiments

proved his germ theory by establishing the fact that different germs came
into the world from their parent germs and germs induce various diseases
which was suggested by earlier workers. He also founded that fermentation
of fruits and grains, resulting in alcohol, was brought about by microbes.
Today the pasteurization process, widely used in fermentation industries, is
the contribution of Pasteur. Pasteur also tackled the problem of anthrax—a
disease of cattle, sheep, and sometimes human beings. In the meantime
Robert Koch (1843-1910) was busy with the anthrax problem in Germany. It
was he who discovered the typical bacilli responsible for the anthrax disease
of cattle and this was the first time a bacterium had been proved to be the
cause of an animal disease.

1.2 KOCH PASTULATE

A series of observations led to the establishment of Koch’s postulates:

(i) A specific organism can always be found in association with a given

disease,

(ii) The organism can be isolated and shown in pure culture in the laboratory,

(iii) The pure culture will produce the disease when inoculated into a
susceptible animal,

(iv) It is possible to recover the organism in pure culture from the

experimentally infected animal.

Again the name of Joseph Lister (1878) is associated with the

concept on pure culture technique. Lister obtained pure cultures of bacteria
and emphasized the importance of growing an organism (bacterium, fungus,
alga, protozoan, or higher forms) in an environment free of any other living
organism, i.e., pure culture. The period from 1880 to 1900 was actually a

4
golden time for microbiology when classical contributions were made leading
to the establishment of microbiology as a science.

The land marks of the history of microbiology are:

(i) Opening of the field of soil microbiology in the late 1880 by the Russian
Serge Winogradsky;

(ii) Application pure culture technique in industrial fermentation by Emil

Christian Hansen (1842-1909) of Denmark, Adametz (1889) of Austria, and
H. W. Conn of the U.S.A. and H. Weigmann of Germany;

(iii) In 1888 the symbiotic relationship between bacteria and leguminous

plants was demonstrated by Hellriegel and Wilfarth;

(iv) A famous Dutch microbiologist, Beijerinck (1851-1981) described the

usefulness of free-living nitrogen-fixing bacteria (Azotobacter) in promoting
soil fertility; and

(v) In the late nineteenth century Burrill, an American scientist working on fire
blight of pears established that bacteria cause plant diseases.

Of late, microorganisms have been used as ideal tools to carry out

the study of intricate life processes. This has inspired physicists, geneticists,
chemists, and biologists to join with microbiologists in what is known as
molecular biology. Mention may be made of some of the contributors and
their contributions in microbiology and molecular biology during the period
from 1944 to 1975: Avery and associates (1944)—DNA carries genetic
information in pneumococcus; Fritzhipman and Hanskrebs (1953) —
physiology and metabolism of living cells; Joshua Lederberg, George
Beadle, and Edward Tatum (1958)—genera in bacteria and their
recombination.

Ochoa and Kornberg (1959) isolated and synthesized RNA and DNA:
Robert W. Holley, Har Govind Khorana and Marshall W. Nirenberg (1968)—
study of genetic code and its function in protein synthesis; Salvador E. Luria
(1969)—functions of organisms in terms of molecular structure including
elucidation of enzyme structure and mode of action; Gerald M. Edelman and

5
Rodney R. Porter (1972)—chemical structures of antibodies; Renato
Dulbecco; Howward M. Temin, and David Baltimore (1975) — enzymes in
RNA tumour virus particles. Microbiologists are also engaged in making
valuable contributions in medical science, industry, agriculture and in
science in general for the welfare of the human society. The basic
knowledge of molecular biology and genetics accumulated during the past
few decades is rapidly being translated into practical objectives and is
revolutionizing industrial microbiology. The most outstanding current
development in applied microbiology is the ability to alter an organism’s
genetic makeup which is commonly referred to as genetic engineering which
area of scientific contribution holds great potential for production of drugs
and vaccines, for improvement of agricultural crops, and various other areas.

1.3 PASTEUR'S EPIC EXPERIMENTS

What was his experimental method? To offset the argument that air
was necessary for spontaneous generation, Pasteur allowed the free
passage of air, but prevented the entry of microbes. He boiled meat broth in
a flask & then drew out & curved the neck of the flask in a flame. No
microbes developed in the flask. When he tilted the flask so some broth
flowed into the curved neck & then tilted it back so the broth was returned to
the base of the flask, microbes grew. Gravity had caused the microbes that
had entered the flask in air & dust to settle at the low point of the neck, never
reaching the broth in the base until the broth washed them in. Pasteur's
success was partly due to good luck. He used meat, which contains few
bacterial endospores (endospores are resistant to heat; many experiments
done prior to Pasteur's used vegetable broths - plants contains many
endospore-forming bacteria.)

1.4 SCOPE OF MICROBIOLOGY

i. Microorganisms and Principles of Biology

Microorganisms help understanding the various principles of biology

as they consist of many characteristics which make them ideal for the
investigation of important biological phenomena. The microbial metabolism

6
follows almost the same patterns that occur in higher groups of organism.
We can study the metabolic patterns of microorganisms and other life
processes at different stages of their growth and reproduction very easily in
comparison to higher organisms. It is because of the fact that the
microorganisms require less space and can be conveniently grown in test-
tubes or flasks; they grow rapidly and reproduce at an unusually high rate
(e.g., certain bacteria reproduce within 20 minutes).

ii. Medical Microbiology and Immunology

Medical microbiology deals with disease producing organisms in

human beings whereas immunology deals with the defense the body puts up
against the pathogens, the disease causing microorganisms, and the factors
which explain resistance to disease. The medical microbiology and
immunology furnishes the basic knowledge on which depend the practical
methods employed for the laboratory diagnosis and prevention of microbial
diseases. It, therefore, gives us a sound foundation for the intelligent
promotion of both, the individual and the public health.

iii. Soil Microbiology:

Soil microbiology deals with the microorganisms present in, and their
role in soil. The most important function of soil microorganisms is to
decompose various kinds of organic matter. Second is the process of
mineralization of various organic constituents. Mineralization of organic
carbon, nitrogen, phosphorus and sulphur via respective cyclic alterations by
soil microorganisms makes these elements available for reuse by plants and
other organisms. As we know, microbes improve the fertility of soil by fixing
atmospheric nitrogen into nitrogen-compounds which are readily used by
plants to synthesize protein and other complex organic nitrogenous
compounds. It is generally true, however, that more the microorganisms
there are in the soil, the more productive it is.

iv. Industrial Microbiology:

Industrial microbiology is that branch of microbiology which deals

with the utility of microorganisms in industrial production of medicines, food

7
supplements, alcohols, beverages, organic acids, vitamins, enzymes etc.
Probably the most significant industrial use of microorganisms was the
production of antibiotics, the wonder drugs. However, some 40 years ago
only a small group of microorganisms were conveniently referred to as
industrially useful microorganisms. But today it is realized that every
microorganism has its own industrial importance. The commercially
beneficial activities of a large number of bacteria, yeasts, molds, and algae
are being exploited, or deserve to be exploited to obtain valuable products.

1.5 Branches of Microbiology

Microbiology is not a mere study of the structural diversity and

classification of microbes, but encompasses the whole gamut of microbial
life. The knowledge of the various aspects of microbes has been
accumulating since the last century and has become so vast that no
microbiologist can claim familiarity with all aspects of the subject.

Microbiological study can be divided basically into the following

branches:

i. Aero-microbiology:

Dispersal of disease causing microbes through air, microbial

population in air, their quality and quantity in air comes under the preview of
this branch.

ii. Agricultural Microbiology:

In this branch, the role of microbes in agriculture is studied from the

point of view of both harm and usefulness. Many microbes—fungi, bacteria
and viruses—cause a number of plant diseases. From the point of view of
benefit—N2 fixing activity, use of microbes as bio -fertilizers and several
other aspects are studied.

iii. Aquatic Microbiology:

Microbiological examination of water, water purification, biological

degradation of waste are studied in this branch.

8
iv. Bacteriology:

This is the largest group among microbes not only in number but also
in importance. Bacteria of both kinds—eubacteria and cyanobacteria (also
known as blue green algae)—are studied here. Bacteria have a profound
influence on various human endeavours including health, industry,
agriculture, etc.

v. Biotechnology:

This is the most significant branch which may even change the
course of life as we know today. Microbes are used as gene carriers to
deliver specific genes to function in a different environment. New, genetically
engineered microbes can produce drugs (human insulin), or in agriculture-
N2 fixing ability may be transferred to all the plants. The potentialities of
biotechnology are immense.

vi. Environmental Microbiology:

This is one of the most important branches of microbiology. The role

of microbes in maintaining the quality of the environment is studied in this
branch. Microbial influence in degradation and decay of natural waste; their
role in biogeochemical cycles are all studied.

Some of the recent researches have shown that certain bacteria can help in
cleaning the oil spill, and this gives added significance to the study of
environmental microbiology.

vii. Food and Dairy Microbiology:

Various aspects such as food processing, food preservation,

canning, pasteurization of milk, study of food borne microbial diseases and
their control is studied.

viii. Geochemical Microbiology:

Role of microbes is coal, gas and mineral formation, prospecting for

coal, oil and gas and recovery of minerals from low grade ores using
microbes, is included here.

9
ix. Immunology:

Studied in this branch are the immune responses in organisms. How

toxins are produced? How the antigens influence the formation of
antibodies? How protective vaccination helps in combating the diseases?
How immune system collapses (as in AIDS), are some of the questions for
which immunology as a branch of microbiology is trying to find out answers.

x. Industrial Microbiology:

The role of microbes in Industrial Production is studied here. Many

microbes produce industrial alcohols and acids as a part of their metabolism.
The study of fermentation by microbes has contributed a lot to alcohol
manufacturing. Breweries have greatly benefitted by understanding the role
of specific microbes in fermentation.

xi. Medical Microbiology:

This branch deals with the pathogenic microbes—their life-cycle,

physiology, genetics, reproduction, etc., Many of the microbes also provide
remedies for microbial diseases. All these aspects are studied in this branch.
Some of the diseases like tuberculosis, leprosy, typhoid, etc. are caused by
microbes, and cure for them is provided by other microbes in the form of
antibiotics.

xii. Mycology:

The study of eukaryotic, achlorophylous organisms generally referred

to as fungi is included in this branch. Some of the common fungi are yeasts,
moulds, mushrooms, puffballs, etc. Fungi are not only harmful but beneficial
also.

xiii. Phycology:

Deals with the study of autotrophic eukaryotic organisms. Members

are generally called algae. Algae include both microscopic as well as
macroscopic members. Only the microscopic algae are studied as a part of
microbiology.

10
xiv. Protozoology

Study of Protozoans in all their aspects comes under the preview of

protozoology. Protozoans are known to cause many diseases like malaria,
amoebic dysentery, sleeping sickness, etc.

xv. Virology:

Viruses are neither eukaryotic not prokaryotic. In fact, they are on the
border line between living and non-living. Viruses cause disease to plants
and animals including human beings. The dreaded AIDS is also caused by a
virus.

LET US SUM UP
In this lesson we studied about the history and the concepts of
microbiology.

CHECK YOUR PROGRESS

1. The name of ---------------- is associated with the concept on pure
culture technique
2. Pasteurization is a method developed by ------------
3. -------------------- deals with disease producing organisms in human

GLOSSARY
1. Microbiology – Study of microbes
2. Bacteriology – Study of Bacteria

SUGGESTED READINGS
1. Microbiology - Pelczar et al
2. Microbiology by Prescott

ANSWERS TO CHECK YOUR PROGRESS

1. Joseph lister
2. Luis Pasteur
3. Medical Microbiology

MODEL QUESTIONS
1. What is Pasterurization

11
UNIT - 2

HISTORY OF MICROSCOPE
STRUCTURE

Overview

Learning Objectives
2.1 Timeline of Microscope
2.2 Types of Microscope
Let us Sum up
Check your Progress
Glossary
Suggested Readings
Answers to check your progress
Model questions

OVERVIEW
Microscope is an instrument to observe te structure and shape of
microbes. The development of microscope is a 600 year process. The
current advanced microscope is having 600 years of history.

LEARNING OBJECTIVES
To know about microscopes and its functions

2.1 TIMELINE OF THE MICROSCOPE

14th century: spectacles first made in Italy

1590: Two Dutch spectacle-makers and father-and-son team, Hans and

Zacharias Janssen, create the first microscope.

1667: Robert Hooke's famous "Micrographia" is published, which outlines

Hooke's various studies using the microscope.

12
1675: Enter Anton van Leeuwenhoek, who used a microscope with one
lens to observe insects and other specimen. Leeuwenhoek was the first to
observe bacteria. 18th century: As technology improved, microscopy
became more popular among scientists. Part of this was due to the
discovery that combining two types of glass reduced the chromatic effect.

1830: Joseph Jackson Lister discovers that using weak lenses together at
various distances provided clear magnification.

1878: A mathematical theory linking resolution to light wavelength is

invented by Ernst Abbe.

1903: Richard Zsigmondy invents the ultramicroscope, which allows for

observation of specimens below the wavelength of light.

1932: Transparent biological materials are studied for the first time
using Frits Xernike's invention of the phase-contrast microscope.

1938: Just six years after the invention of the phase contrast microscope
comes the electron microscope, developed by Ernst Ruska, who realized
that using electrons in microscopy enhanced resolution.

1981: 3-D specimen images possible with the invention of the scanning
tunneling microscope by Gerd Binnig and Heinrich Rohrer.

Origin: The origin of the word microscope according to the Online

Etymology Dictionary is as follows: 1656, from Mod.L. microscopium, lit. "an
instrument for viewing what is small," from Gk. micro- (q.v.) + -skopion.
"means of viewing," from skopein "look at." Microscopic "of minute size" is
attested from 1760s.

2.2 TYPES OF MICROSCOPE

1. Simple microscope

2. Compound microscope

3. Electron microscopes

4. Phase-Contrast microscope

13
 5. Interference microscope

1. Simple Microscopes:

Simple/ Dissecting Microscope:

As shown in the figure, dissecting microscope consists of a biconvex

lens which is moved up and down by an adjustment screw to bring the object
in sharp focus. The object is placed on the platform and light is focused with
the help of a concave mirror fitted below. In simple microscope, convex lens
of short focal length is used to see magnified image of a small object. The
object is placed between the optical centre and the focus of a convex lens,
its image is virtual, erect and magnified and on the same side as the object.
The position of the object is so adjusted that the image is formed at the least
distance of distinct vision (D).

Magnifying power (M) of a simple microscope is the ratio of the angle

subtended by the image at the eye to the angle subtended by the object
seen directly, when both lie at the least distance of distinct vision or the near
point.

M = 1 + D/ f

Where D is the least distance of distinct vision and f is the focal length of the
lens.

14
 Fig. 2.1 Simple Microscope

2. Compound Microscope:

A compound microscope consists of two set of convex lenses. A lens

of short aperture and short focal length facing the object is called objective.
Another set of lens of relatively moderate focal length and large aperture
facing the eye is called the eye piece. The objective and the eye piece are
placed coaxially at the two end of a tube. The object is placed between the
centre of curvature and focus of the objective – it forms real, inverted and
magnified image on the other side of the objective. This image acts as an

15
object for the eye piece which then acts as a simple microscope to produce
virtual, erect and magnified image.

Magnifying power (M) of a compound microscope will be

M = L/ f0 (1+D/fe)

Where f0and fe are focal length of objective and eye piece respectively, L is
the length a the microscope tube and D is the least distance of distinct
vision.

Fig. 2.2 Compound Microscope

16
3. The Electron Microscope:

The organelles of the cell became known after the electron

microscope was invented. The electron microscope was developed in 1932
by M. Knoll and Ruska in Germany. It consist of a source of supplying, a
beam of electron of uniform velocity, a condenser lens for concentrating the
electron on the specimen, a specimen stage for displacing the specimen
which transmits the electron beam, an objective lens, a projector lens and a
fluorescent screen on which final image is observed.

For permanent record of the image, the fluorescent screen is

replaced by photographic film. This microscope utilizes a stream of high
speed electrons which are deflected by an electromagnetic field in the same
way as a beam of light is reflected when it crosses a glass lens. There are
two types of electron microscope.

Fig. 2.3 Electron Microscope

17
 Fig. 2.4 Schematic Diagram of A-TEM; B-TEM

General Principle of EM

The fundamental principle of EM is similar to those of LM. In EM, a

high velocity beam of electrons (instead of light) is used to travel in a
vacuum tube. The beam of electrons is focused by a series of
electromagnetic lenses analogous to the condenser, objective and eye piece
lenses of the light microscope. The object is placed between the condenser
and objective. The magnified image of the object is formed on the
fluorescent screen or on photographic film rather than being observed
through eye piece. Since the image produced by electrons does not have the
colour, the electron micrograph always has shades of black, grey and white.
The objects under examination must be extremely thin and are treated with
chemicals or dyes to enhance the contrast as such the live objects cannot be
studied. Techniques like negative staining, shadow casting and tracers are
commonly used to increase the contrast.

Theoretically, the maximum resolution of the EM is 0.005 nm which is

less than the diameter of a single atom, or 40,000 times the resolution of the
light microscope and 2 million times that of the naked eye. However, the
practical resolution of modern EM is of 0.1 nm (1 A).

18
(a) Transmission electron microscope (TEM)

This is used to observe fine structure of cells. Ultra thin sections of

the object are prepared and they are stained with a heavy metal (gold or
palladium) to make certain part dense, and inserted in the vacuum chamber
of the microscope. A 100, 00 volt electron beam is focused on the section
and manipulated prepared from the image may be enlarged with enough
resolution to achieve a total magnification of over 20 million times.
Transmission electron microscope and scanning electron microscope. The
other improved relatives of EM are scanning probe microscope, scanning
tunneling microscope and atomic force microscope.

The TEM was designed by Knoll and Ruska (1932) of German. The
TEM has a magnification of 100,000-300,000 times with resolution of 2-5 A.
The resolution of TEM depends upon wave length (A) of the electron beam.
The wave length of electron beam is inversely proportional to the square root
of the accelerating voltage (V) i.e. λ /V. Electron beam produced by an
electric current of 50,000 Volts (V) has a wavelength of a electron is 0.5 A. It
is less than the diameter of smallest atom (Hydrogen atom = 1.06(A). A
wavelength of 0.5 A shall produce a resolution power of about 0.25 A.

The same is not achieved due to limitations of:

(i) Electromagnetic lenses,

(ii) Drying up of specimens,

(iii) Sectioning of specimens,

(iv) Creation of perfect vacuum.

19
Structural parts of a TEM- The structural parts of a TEM are as follows

Fig. 2.5 TEM

(a) Electron gun

It consists of a tungsten filament or cathode that emits electrons on

receiving high voltage electric current (50,000-100,000 volts). Near the top of
the tube is an anode which attracts electrons.

(b) Ray tube (Microscope Column)

It is a high vacuum metal tube (2mt. high) through which electrons travel.

(c) Condense lens

It is the electromagnetic coil which focuses the electron beam in the plane of
the specimen.

(d) Objective lens

It is the electromagnetic coil which produces the first magnified image

formed by the objective lens and produces the final image.

20
(e) Projector lens

It is also an electromagnetic coil which further magnifies the first

image formed by the objective lens and produces the final image. Each
electromagnetic coil has a coil of wire encased by a soft iron casing.

(f) Fluorescent Screen or Photographic Film

Since unaided human eye cannot observe electrons, therefore, a

fluorescent screen is used for viewing the final image of the specimen. The
final image can be captured on photographic film and die photograph
obtained is called an electron micrograph.

Uses

1. EM has a high magnification and resolving power.

2. Without the aid of EM, biologists would have never known submicroscopic
cell organelles (e.g., ribosomes, micro-bodies, centrioles, microtubules,
endoplasmic reticulum) and internal structure of microscopic organelles
(e.g., chioroplasts, mitochondria).

3. Study of microorganisms, viruses and viroids have been possible only with
the aid of EM.

4. It can discern even macromolecules.

This has helped scientists to know the arrangement of molecular aggregates

and even their components, e.g. nucleosomes.

Draw backs:

(i) It is complicated and costly.

(ii) There is risk of radiation leak,

(iii) 11 require very high voltage electric current.

(iv) A cooling system is required,

(v) The specimen or object has to be given special treatment including

complete dehydration.

(2) Scanning Electron Microscope (SEM)

21
 It is the second type of EM, first built by Knoll (1935) but it was
commercially developed by Cambridge Instruments (1965). It is used to
study the three dimensional images of the surfaces of cells, tissues or
particles. The SEM allows viewing the surfaces of specimens without
sectioning. The specimen is first fixed in liquid propane at-180° C and then
dehydrated in alcohol at-70°C. The dried specimen is then coated with a thin
film of heavy metal, such as platinum or gold, by evaporation in a vacuum
provides a reflecting surface of electrons. The surface of the specimen when
scanned by electron beam release secondary electrons that from a three-
dimensional image of the specimen on a television screen. Holes and
fissures appear dark, and knobs and ridges appear light. Complete scanning
from top to bottom usually takes only a few second.

Fig. 2.6 Principle of SEM

It is used to study the surfaces of the cell and organisms. In this

microscope, the image is formed by electrons reflected back from the object.
The image formed by this microscope has a remarkable three dimensional
appearance. Typically magnification of scanning electron microscope is
around 20,000 times.

4. Phase-Contrast microscope

This is used to study the behavior of living cells, observe the nuclear
and cytoplasmic changes taking place during mitosis and the effect of

22
different chemicals inside the living cells. By using the phase-contrast
microscope, an image of strong contrast of the object is obtained. It is a
contrast-enhancing optical technique that can be utilized to produce high-
contrast images of transparent specimens, such as living cells (usually in
culture), microorganisms, thin tissue slices, fibers, glass fragments, and sub-
cellular particles (including nuclei and other organelles). In effect, the phase
contrast technique employs an optical mechanism to translate minute
variations in phase into corresponding changes in amplitude, which can be
visualized as differences in image contrast.

One of the major advantages of phase contrast microscopy is that

living cells can be examined in their natural state without previously being
killed, fixed, and stained. As a result, the dynamics of ongoing biological
processes can be observed and recorded in high contrast with sharp clarity
of minute specimen detail.

Fig. 2.7 Phase Contrast Microscope

5. Interference microscope:

Interference microscope is used for quantitative studies of

macromolecules of the cell components, for example it is used for
determination of lipid, nucleic acids and protein contents of the cell.

23
Interferometry is a traditional technique in which a pattern of bright and dark
lines (fringes) result from an optical path difference between a reference and
a sample beam.

The incoming light is split inside an interferometer, one beam going

to an internal reference surface and the other to the sample. After reflection,
the beams recombine inside the interferometer, undergoing constructive and
destructive interference and producing the light and dark fringe pattern.

A precision translation stage and a CCD camera together generate a

3D interferogram of the object that is stored in the computer memory. This
3D interferogram of the object is then transformed by frequency domain
analysis into a quantitative 3D image providing surface structure analysis.

Fig. 2.8 Interference Microscope

LET US SUM UP
In this unit we studied the history and the structure and functions of
microscopes in detailed.

CHECK YOUR PROGRESS

1. Who created the first microscope –------------

24
 2. Robert Hook published his work as ------------

3. Electron Microscope was developed by -------

GLOSSARY
1. TEM – Transmission Electron Microscope

2. SEM – Scanning Electron Microscope

SUGGESTED READINGS

1. Microbiology by Pelczar et al

ANSWERS TO CHECK YOUR PROGRESS

1. Hans and Zacharias Janssen

2. Micrographia

3. M. Knoll and Ruska

MODEL QUESTIONS
1. Explain Compound Microscope

25
UNIT - 3

CLASSIFICATION AND ULTRA

STRUCTURE OF BACTERIA
STRUCTURE

Overview

Learning Objectives
3.1 Five Kingdom Classification
3.2 Classification of Bacteria by Bergey
3.3 Ultra Structure of Bacterial Cell
Let us Sum up
Check your Progress
Glossary
Suggested Readings
Answers to check your progress
Model questions

OVERVIEW
Microbes are classified by many scientists wit the various parameters
like shape structure, cellwall content habitat physical and chemical
properties. The structure involves the chemical composition of cell wall and
its functions.

LEARNING OBJECTIVES
To understand the microorganisms and its classes

To know the structure of the bacteria.

26
3.1 FIVE-KINGDOM SYSTEM OF CLASSIFICATIONS

Prokaryotic and eukaryotic organisms were distinguished on the

basis of cell anatomy, and the concept of a bacterium as a prokaryotic
organism was established in microbiology in 1962 by Stamir and Van Niel. In
1969, Whittaker proposed a five kingdom system consisting of kingdom of
plantae, fungi, animalia, protista and monera for all organisms on the basis
of their energy- yielding systems and cell anatomy.

Microorganisms with the common characterstics described above are

distributed in the kingdoms of monera, protista, fungi and a part of plants.
Recently, evolutionary relationships of living organisms have been defined
on the basis of ribosomal RNA sequences and other data.

Fig. 3.1 Wittaker Five Kingdom Classifiation (1969)

27
The kingdom Monera of prokaryoteae includes all prokaryotic
microorganisms. Protista consists of unicellular or multicellular eukaryotic
organisms but true tissues are lacking. The kingdom Fungi contains
eukaryotic and multinucleate organisms. The members have absorptive
mode of nutrition. Animalia contains multicellular animals devoid of cell wall.
Ingestion is the mode of nutrition. The kingdom Plantae includes multicellular
eukaryotes. Their mode of nutrition is the photosynthesis.

3.2 CLASSIFICATION OF BACTERIA BY BERGEY (1969)

Bergey (1969) Classified Bacteria into 19 groups, viz,

1. Phototrophic Bacteria: Rhodospirillum - Rhodopseudomonas –

Chromatium

2. Gliding Bacteria: Myxococcus - Beggiatoa - Simonsiella - Leucothrix

3. Sheathed Bacteria: Sphaerotilus - Leptothrix

4. Budding / Appendaged Bacteria: Caulobacter - Gallionella

5. Spirochetes: Spirochaeta - Treponema - Borrelia

6. Spiral and Curved Bacteria: Spirillum - Auqaspirillum - Oceanospirillum -

Bdellovibrio

7. Gram-negative Aerobic Rods and Cocci: Pseudomonas -

Xanthanomonas - Zoogloea - Gluconobacter - Azotobacter - Rhizobium -
Agrobacterium - Halobacterium - Acetobacter

8. Gram-Negative Facultative Anaerobic Rods: Escherichia - Citrobacter -

Salmonella - Shigella - Klebsiella - Enterobacter - Serratia - Proteus -
Yersinia - Erwinia - Vibrio - Aeromonas - Zymomonas - Chromobacterium -
Flavobacterium -

9. Gram-negative anaerobes: Bacteriodes - Fusobacterium - Desulfovibrio

- Succinimonas

28
10. Gram-Negative cocci: Nisseria - Branhamella - Acinetobacter -
Paracoccus

11. Gram-negative anaerobic cocci: Veillonella - Acidaminococcus

12. Gram-Negative Chemolithotrophic: Nitrobacter - Thiobacillus -

Siderocapsa

13. Methane producing

14. Gram-Positive Cocci: Micrococcus - Staphylococcus - Streptococcus -

Leuconostoc - Pediococcus - Aerococcus - Peptococcus - Ruminococcus -
Sarcina

15. Endospore-forming Rods and cocci: Bacillus - Clostridium -

Sporosarcina

16. Gram-positive, non-sporing rods: Lactobacillus - Listeria -

Erysipelothrix - Caryophanon

17. Actinomycetes and Related: Corynebacterium - Arthobacter -

Brevibacterium - Cellumonas - Kurthia - Propionibacterium - Eubacterium -
Actinomyces - Archina - Bifidiobacterium - Rothia - Mycobacterium - Frankia
- Streptosporangia - Nocardia - Streptomyces - Streptoverticillium -
Micromonospora

18. Rickettsias: Rickettsia - Erhlichia - Wollbachia - Bartonella - Chlamydia

19. Mycoplasmas: Mycoplasma - Acoleplasma - Thermplasma –

Spiroplasma

3.3 ULTRA STRUCTURE OF BACTERIAL CELL

Let us learn about Morphology and Ultrastructure of a Bacterial Cell.

After reading this article you will lean about: 1. Size of a Bacterial Cell 2.
Shape and Arrangement of Bacterial Cell 3. Ultra-structure of Bacterial Cell
4. Structures Outside the Cell Membrane 5. Structure of Cell Membrane 6.
Cell Envelope of Prokaryote 7. Bacterial Cell Wall: Peptidoglycan
Component and Other Details.

29
Size of a Bacterial Cell

There are great variations in size of bacteria. They measure from

0.75 µ to 1.5 µ but on an average each cell of bacterium measures about
1.25 µ to 2 µ (a in diameter. Some times their size varies due to slide
preparation. The smallest rod shaped eubacteria is Dialister Pneum osintes
and measures between 0.15 µm to 3.0 µm size. Sulphur bacteria, Thiophysa
volutans has diameter of about 18 µm and is considered as largest amongst
all bacteria. Bacteria are so minute that a single drop of water may contain
about billions of bacteria.

Shape and Arrangement of Bacterial Cell

Bacterial cells exhibit greater variation in their shape but usually four
conventional shapes of cell have been recognized
1. Cocci
Simplest form of bacteria in which bacterium appears like a spherical cell.
(i) Micrococci
When bacterium appears singly.
(ii) Diplococci
When they appear in pairs of cells.
(iii) Streptococci
When they appear in chain form.
(iv) Tetrad
Arranged in square of four.
(v) Sarcinae
When arranged in cuboidal or in different geometrical or packet
arrangement.
(vi) Staphylococci
Arranged in irregular clusters like bunch of grapes.

30
 Fig. 3.2 Shapes of Bacteria

2. Rod Shaped
They are also called bacilli and are commonest in microbial world.
They are of two kinds
(i) Short rods:
Very short rods, occurring mostly singly.
(ii) Long rods
Cylindrical shape, are known as Bacilli or rods, occurring singly, in pairs or in
chains.

31
3. Vibrios
They are curved rods or comma shaped, their curvature is always less than
a half turn.
4. Spirilla
They are curved or spiral shaped cells, their curvature exceeds that
of a half turn. They may be classified as either spirilla or spirochetes. Few
bacteria actually are flate. For instance, Anthony E. Walsy has discovered
square bacteria living in salt ponds. These bacteria are shaped like flate,
square to rectangular box about 2 µm to 4 µm and only 0.27 µm thick.
However, some bacteria are variable in shape and have a single
characteristic form. These are called Pleomorphic (corynbacterium,
Arthrobacter).
3.3 Ultra-Structure of Bacterial Cell

Examination of bacterial cells with electron microscope reveals

various component structures. Some of these are outside the cell
membrane; others are internal to cell membrane.

Fig. 3.3 Ultra structure of Bacterial Cell

Structures Outside the Cell Membrane:

Capsule:

32
 Some prokaryotic organisms secrete slimy or gumy materials
(exopolymers) on their surface. A variety of these structures consist of
polysaccharides and a few consist proteins. The more general term
glycocalyx is also used. The glycocalyx is defined as the polysaccharide
containing material lined outside the cell. Composition of these layers varies
in different organisms but can contain glycoprotein and different
polysaccharides including polyalcohol and amino sugars. These layers may
be thick, or thin, and rigid or flexible, depending on heir chemical nature in
specific organism. The rigid layers are organized in a tight matrix that
excludes Indian ink, this form is referred to Capsule.

Most bacterial capsules are composed of polysaccharides. Capsules

composed of single kind of sugar are termed homopolysaccharides; are
usually synthesized outside the cell from disaccharides by exocellular
enzyme. The synthesis of glucan from sucrose by S. mutans. other capsules
are composed of several types of sugar and are termed as
heteropolysaccharides, for example, capsule of Klebsiella pneumoniae. A
few capsules are polypeptides. For example B. anthracis, is composed
entirely of a polymer of glutamic acids.

Functions of Capsule

Capsule is not essential for survival of organism under favourable

growth conditions. It, however, provides protection in unfavourable
environments. Presence of capsules is of importance in disease causing
ability of some bacteria. The non-capsulated strains of Pneumococcus are
destroyed by host phagocytes and are therefore not effective. The
incapsulated strains of the bacterium are protected from phagocytosis and
take part in virulence in addition because outer polysaccharide layer bounds
a significant amount of water so it is resistant to desiccation.

Flagella

Most motile procaryotes move by use of flagella thread like locomotor

appendages, extending outwards from the cell membrane and cell wall.
Bacterial flagella are slender, rigid structures, about 20 nm across and up-to

33
15 to 20 µ m long. Flagella are so thin that they cannot be observed directly
with bright field microscope. But must be stained with special techniques
designed to increase their thickness. Bacteria species often differ
distinctively in their patterns of flagella distribution and these patterns are
useful in identifying bacteria. Monotrichous bacteria have one polar
flagellum. Amphitrichous bacteria have either singe or duster of flagellum at
both pole.

In contrast, Lophotrichous bacteria, have a cluster of flageila at one

ends. Bacteria surrounded by lateral flageila are Peritrichou.

Fig. 3.4 Flagella attachment

Flagellar Ultra-Structure:

Transmission electron microscope studies have shown that flagellar

apparatus is made up of three distinct regions

(1) The outermost region is filament which is extended from the cell surface
to tips.

(2) Basal bodies consist of small central rods inserted into cells

(3) A short curved segment, the flagellar hook links the filament to basal
bodies and acts as flexible coupling.

34
 Fig.3.5 Ultrastruture of Flagella

The filament is a hollow, rigid cylinder made up of protein subunits

flagellin which ranges in molecular weight from 30,000 Dalton to 6,000
dalton, depending on the bacterial species. Hook and basal bodies are quite
different from filaments. Basal body is more complex part of flagellum. In E.
coli and most Gram-negative bacteria, basal bodies bear 2 pairs of ring,
outer pair (L and P ring) is situated at the level of outer membrane and inner
pair (S and M ring) is located near the level of cell membrane. The outer L
and P ring associates with lipopolysaccharides and peptidoglycan layer
respectively. Inner M ring contacts the plasma membrane while S ring lies
just above attached to inner surface of peptidoglycon,. Flageila of Gram-
positive bacteria have only lower S and M ring.

Fimbriae or Pili

Some bacteria mostly (Gram negative bacilli) contain, non-flagellar,

extremely fine appendages called fimbrial or pile. The filament of pilus is
straight and diameter is 7 n m. It is made up of pilin protein. Molecular weight
is 17,000. Pili are nonmotiie but adhesive structure. They enable the bacteria
to stick firmly to other bacteria, to a surface or to some eucaryotic such as

35
mould plants, plants and animal cells including R.B.C and epithelial cells of
elementary, respiratory and urinary tracts. Pilli help in conjugation (e.q. F. pili
or sex pili) of male bacteria, in the attachment of pathogenic bacteria to their
host cell. Pile are known to be coded by the genes of plasmid that determine
cell capacity to carry out conjugative genetic exchange with other cells.

Spinae

Some Gram-positive bacteria have tubular unicellular and rigid

appendage of singe protein moity called spinae. They are known to help the
bacterial ceil to tolerate some environmental conditions such as salinity, pH
and temperature etc.

Structure of Cell Membrane

Cell membrane is a thin structure that completely surrounds the cell

only about 8 nm thick. This structure is critical barrier separating the inside of
cell from environment. The cell membrane is also highly selective barrier
enabling the cell to concentrate a specific metabolite and excrete waste
material. Mostly biological membrane is composed primarily of
Phospholipids (about 20 to 30 percent) and proteins (about 60 to 70
percent). The phospholipids form a bilayer in which most of the proteins are
strongly held (integral proteins) and these proteins can be removed only by
destruction of the membranes, as with treatment by detergents. Other
proteins are only loosely attached (Peripheral proteins) can be removed by
mild treatment such as osmotic shock. The lipid matrix of membrane has
fluidity, allowing the components to move around laterally. Fluidity is
essential for various membrane functions and is dependent on factors such
as temperature and on proportion of unsaturated fatty acids to saturated fatty
acids present in phospholipids.

36
 Fig.3.6 Scematic representation of Lipid bi layer

One major difference in chemical composition of membrane between

eukaryotic cells and prokaryotic cells is that eukaryotes have sterol in their
membrane depending on cell type, sterol can make up from 5-25% of total
lipid of eukaryotic membrane. Sterols are absent from membranes of all
prokaryotic cells (methanotropns are major exception). Sterol are rigid,
planner molecules, whereas fatty acids are flexible. The association of sterol
with membrane serves to stabilize its structure and make it less flexible.
Membrane rigidity may be necessary in eucaryotes because many of them
lack a rigid cell wall. Rigid structure provides stability to the cell. Molecules
similar to sterol called Hopanoid, are present in several bacteria and may
play role similar to that of sterol in eukaryotic cell.

Fig. 3.6 Ester linkage of Eubacteria and Ether linkage Archaebacteria

A significant difference exists between the phospholipids of

eubacteria and those of archaebacteria. In eubacteria the phospholipids are
phosphoglycerides in which straight chain fatty acids acids are ester linked
to glycerole. In archaebacteria, the lipids are polyisoprenoid branched chain
lipids, in which long chain branched alcohol (Phytanols) are ether linked to
glycerol.

37
Cell Envelope of Prokaryote

Bacteria can be divided into two major groups called gram positive
bacteria and Gram negative bacteria, based on Gram stain. Gram positive
bacteria and Gram negative bacteria differ in the appearance of cell wall.
The cell wall of Gram negative bacteria is multilayered structure and quite
complex whereas Gram positive bacteria contain primarily single type of
molecule and is often much thicker.

Fig. 3.7 Schematic diagram of Bacterial envelope

Bacterial Cell Wall: Peptidoglycan Component

In the cell wall of bacteria, there is one rigid layer that is primarily
responsible for strength of the wall. In Gram negative bacteria addition layer
is present outside this rigid layer. The rigid layer of both Gram negative
bacteria and Gram positive bacteria is very similar in chemical composition
and is called Peptidoglycan (or murein). This layer is thin sheet composed of
two sugar derivatives, N-acetyl glucosamine and N-acetyl muramic acid and
small number of amino acids consisting of L-alanine, D-alanine, D-Glutomic
acid and either lysine or meso-diamino palmilic acid acid. These constituents
are connected to form a repeating structure, glycan tetrapeptide. Basic
structure of peptidoglycan is a thin sheet in which the glycon chains formed
by sugars are connected by peptide cross links formed by the amino acid.
Glycosidic bonds connecting the sugars in the glycan chains are very strong
but these chains alone cannot provide rigidity in all directions. The full

38
strength of amino acids reliazed only when these chains are cross-linked by
amino acids. In Gram-negative bacteria cross linkage usually occurs by
direct peptide linkage of the amino group of Diamino palmilic acid to the
carboxyl group of the terminal D-alanine. In Gram positive bacteria cross
linkage is usually by peptide inter bridge. The kinds and number of cross
linking amino acids varies from organism to organism.

In Staphylococcus aureus the peptidoglycan is made up of linear

glycon (Polysaccharide) Chains connected through short tetrapeptide and
pentagiycine bridge. The polysaccharide chain consisting of alternate
residues of the amino acid N-acetyl glucosamine (NAG) and N-acetyl
muramic acid (NAM) linked in β-1, 4 glycosidie linkage. Each NAM residue
carries a short peptide chain of four amino acid residues (tetra peptide)
which are L-alanine, D-Glutamic acid, L-Lysine and D-alanine. Tetra peptide
is unusual in that it contains D-amine acid, which is rarely found in protein.
Neighbouring tetrapeptides are linked by pentaglycine bridge peptide, each
consisting of five glycine residues. Pentaglycine bridge peptide links L-lysine
of one tetrapeptide with the terminal D-alanine of its neighbour. Because of
extensive cross linking the cell wall has a rigid structure frame work.

Diversity of Peptidoglycon

Peptidoglycon is present only in bacteria, The sugar N Acetyl

muramic acid and mesodiaminopimelic acid are never found in the cell walls
of Archael and eukaryote. However, not all bacteria have DAP in their
peptidoglycan. DAPA is present in all gram negative bacteria and in some
gram postive bacteria but most Gram +ve cocci have lysine instead of DAP
and a few other Gram positive bacteria have other amino acids. In
peptidoglycan, The glycon portion is uniform with only the sugar NAG and
NAM being present, and these sugars are always connected in p, 1→ 4,
linkage. Tetra peptide of the repeating unit shows major variation in only one
amino acid, the Lysine, diaminopimetic acid alteration. However, the D-
Glutamic at position 2 can be hydroxylated in some organisms, whereas
substitution occurs in amino acids at positions 1 and 3 in a few others.

39
 More than 100 different peptidoglycan types are known and the
greater variation among them occurs in inter-bridge. Any of the amino acids
present in the tetra peptide can also occur in the inter bridge, but in addition,
a number of other amino acids can be found there such as glycine,
threonine, serine and a aspartic acid. Branched chain aminoacids, aromatic
amino acids, sulphur containing amino acids and histidine, arginine and
proline are never found in the inter bridges.

Teichoic Acids

Gram positive bacteria have acidic polysaccharide called teichoic

acids attached to their cell wall. The term teichoic acid includes all wall,
membrane or capsular polymers containing glycerophosphate or ribitol
phosphate residuces. These poly-alcohols are connected by phosphate
ester bond to 6-OH group of NAM. Because they are negatively charged,
Teicohic acids are partially responsible for the negative charge of cell
surface and may function to effect passage of ions through the cell wall.
Certain Glycerol containing acids are bound to membrane lipids of Gram
positive bacteria, because these teichaic acids are intimately associated with
lipid, they have been called lipo-teichoic acid.

Fig. 3.8 Teichaic acid

Protoplast Formation

Peptidoglycass, can the signature molecule of bacteria, can be

destroyed by lysozyme, a protein that breaks the β, 1-4 glycosidic bonds

40
between N-acetyl glucosamine and N-acetyl muramic acid in peptidoglycan;
thereby weakening the bond. Water then enters the cell and the cell swells
and eventually bursts, a process called lysis. Lysozyme is found in animal
secretion including tears, saliva and other body fluids and functions as major
line of defence against infection by bacteria.

Fig. 3.9 Lysis

If the proper concentration of a solute that does not penetrate the cell, such
as sucrose is added to the medium, the solute concentration out-side the cell
balances that inside. Under these conditions, lysozyme digests
peptidoglycan, butt lysis does not occur, and instead a protoplast is formed.

Fig. 3.10 Cell Lysis

Pseudopeptidoglycan and other Cell Walls of Archae Bacteria

Certain Archae contain cell walls constructed of a polysaccharide

very similar to peptidoglycon. This material is called Pseudopeptidoglycan.
The backbone of Pseudopeptidoglycan is composed of alternating
repeats of N-acetyl glycosomine and N-acetyltalosaminuric acid. The

41
back bone of Pseudopeptido-glycan also varies from peptidoglycan in that
glycosidic bonds are 1, 3 instead of 1, 4, found in true peptidoglycon, e.g.,
Methanobacterium Cell walls of other Archae lack both Peptidoglycan and
Pseudopeptidoglycan and consist of Polysaccharide, glycoprotein or protein.

Fig. 3.11 Pseudopeptidoglycon

Methanosarcina species (Methanogenic Arche) contain thick polysaccharide

wall composed of glucose, glucuronic acid, galactosamine and acetate.
Extreme halophilic (salt loving) Archea such as holococcus produce walls
similar to those of Methanosarcina but also contain abundance of sulphate
(SO-42) residue. However, the most common wall type among archae
bacteria is the paracrystaline surface layer (S-layer) consisting of proteins or
glycoproteins, generally of hexagonal symmetry S-layer have been found
among species of all groups of Archae, the extreme halophile, the
methianogens and the higher hermophiles Except Thermoplasma (55°C
sp H = 2), all archae contain a cell wall and as in bacteria, the archaeal cell
wall functions to prevent osmotic lysis and to define cell shape. Due to
absence of peptidoglycon in cell wall, all archae naturally resistant to action
of lysozyme and penicilline agent that destroys this molecule.

Outer Membrane of Gram Negative Bacteria

42
 In Gram negative bacteria outer membrane is covered by
lipopolysaccharide. The lipopolysaccharide consists of complex
polysaccharide covalently linked to lipid A. Polysaccharide consists two
portions, the core polysaccharide and O-polysaccharide. In salmonella, the
core polysaccharides consists of ketodeoxyoctonate (KDO), seven carbon
sugar heptose, glycose, galactose and N-acetyl Glucosamine. Connected to
the care is the O-polysaccharide which usually contains galactose,
Rhamnose, manose (six carbon) and dideoxy sugar abequose. These
sugars are connected in repeating sequence. Lipid portion of
lipopolysaccharide, referred to as lipid A is not a glycerol lipid, but instead
the fatty acids are connected by ester amine linkage to a disaccharide
composed of N-acetyl glucosamine phosphate. The disaccharide is attached
to core-o-polysaccharide through KDO. Fatty acid commonly found in lipid A,
lauric, muristic, Palmitic and β-hydroxy meristic acid acid.

Fig.3.12 Schematic representation of outer wall of Gram Negative

Bacteria
In outer membrane, the LPS associated with various proteins to form outer
half of unit membrane sheet. Alipo protein complex is found on inner side of
the outer membrane of Gram negative bacteria, lipo protein is small protein
that functions as an anchor between the outer membrane and peptidoglycon.

43
Porins:

Outer membrane of Gram negative bacteria is relatively permeable to

small molecules. This is because proteins called porins are present in the
outer membrane of gram negative bacteria and function as channels for the
entrance and exit of hydrophilic low mw substance. Several porin have been
identified, and both specific and non-specific classes are known. Non-
specific porins form water filled channels through which small substance of
any type can pass. Byn contrast, some porins are highly specific because
they contain a specific binding site for one or more substances. Porins are
protein containing three identical sub-unis. Porins are trans-membrane
protein and associate to form small membrane holes about 1 nm in diameter.
Through the action of porins, the outer membrane is permeable to small
molecule. However, O.M. is not permeable to enzyme and other large
molecules. One of the major functions of outer membrane may be to keep
certain enzymes, which are present outside the cytoplasmic membrane, from
diffusing away from cell.

Periplasm

Space between the outer surface of the cytoplasmic membrane and

inner surface of the LPS containing outer membrane is called Periplasm. It is
gel like in consistency, because of the abundance of periplasmic protein.
Periplasm of gram negative bacteria several proteins including hydrolytic
enzyme which functions in initial degradation of food, binding protein which
being the process of transporting substrate and chemoreceptors, which are
protein involved in chemotaxis.

Bio-Synthesis of Peptidoglycan

Peptidoglycan layer can be thought of as stress bearing fabric much

like a sheet of rubber. Synthesis of New peptidoglycan during cell growth
involves control cutting by autolysis of bonds connecting small area of pre-
existing peptidoglycan, with simultaneous insertion of new pieces of
peptidoglycan. This process continues during cell division when cell volume
increases until a cross wall (septum) forms and the cell is divided into two

44
cells. Two carrier molecules participate in peptidoglycan synthesis, uridinidie
phosphate and lipid carrier, called bactoprenol is a 55 isoprenoid alcohol that
is connected via phosphodiester linkage to N-acetyl C-muramic acid to which
a pentapeptide is attached. The second amino sugar of peptidoglycan, N-
acetyl glucose amine is then added followed by the addition of the
pentaglycine bridge.

Building block of new chain is synthesized in the cytoplasm on the

inner surface of cytoplasmic membrane, these are polymerized into
peptidoglycona polymer and attached to lipid carrier bactoprenol
(undocaprenol), which mediate their transport through the cell membrane.
On the outer surface. The monomers, still attached to the membrane through
the bactoprenol carrier, are polymerized into unit peptidoglycan strands that
average about 30 disaccharide in length. These are released into periplasm
and diffuse to a point in the wall, permit entry of new strand into
peptidoglycan layer. There, they are incorporated by cross linking the new
strand to those already present in the wall.

LET US SUM UP
In this unit describes the classification of bacteria and the ultra
structure of bacteria. Peptidoglycan (murein) is an essential and specific
component of the bacterial cell wall found on the outside of the cytoplasmic
membrane of almost all bacteria Its main function is to preserve cell integrity
by withstanding the turgor. Indeed, any inhibition of its biosynthesis
(mutation, antibiotic) or its specific degradation (e.g. by lysozyme) during cell
growth will result in cell lysis. Peptidoglycan also contributes to the
maintenance of a defined cell shape

CHECK YOUR PROGRESS

1. Five kingdom classification proposed by ------------------
2. Most commonest shape in the bacterial world -------------

45
GLOSSARY
1. Tetrad – Bacterial cell arranged in a square
SUGGESTED READINGS
1. Text Book of Microbiology by Jayaram Paniker and Ananthanarayan

ANSWERS TO CHECK YOUR PROGRESS

1. Whittaker

2. Bacilli

MODEL QUESTIONS
1. Write detailed notes on Bacterial cell wall

46
UNIT - 4

BACTERIAL STAINING
STRUCTURE

Overview

Learning Objectives
4.1 Simple Staining
4.2 Differential Staining
Let us Sum up
Check your Progress
Glossary
Suggested Readings
Answers to check your progress
Model questions

OVERVIEW
Most types of cells do not have much natural pigment and are
therefore difficult to see under the light microscope unless they are stained.
Several types of stains are used to make bacterial cells more visible. In
addition, specific staining techniques can be used to determine the cells’
biochemical or structural properties, such as cell wall type and presence or
absence of endospores.

LEARNING OBJECTIVES
To understand and identify the bacteria strain by staining the
bacterial cell with different type of stains.
4.1 Simple Staining Procedure

When a single staining-reagent is used and all cells and their

structures stain in the same manner, the procedure is called simple staining
procedure.

47
 This procedure is of two types – positive and negative. In
positive staining, the stain (e.g., methylene blue) is basic (cationic)
having positive charge and attaches to the surface of object that is
negatively charged.

Outline of Bacterial staining

In negative staining, the stain (e.g., India ink, nigrosin) is acidic

(anionic) having negative charge and is repelled by the object that is
negatively charged, and thus fills the spaces between the objects
resulting in indirect staining of the object.

Fig. 4.1 Simple Staining

4. 2 Differential Staining Procedure

When more than one staining reagents are used and specific
objects (e.g., specific microorganisms and/or particular structure of a

48
microorganism) exhibit different staining reactions readily
distinguishable, the procedure is called differential staining. The most
widely used differential staining in microbiology are Gram-staining and
acid-fast staining. Acid-fast staining is especially useful in identifying
Mycobacterium tuberculosis, the causative agent of tuberculosis.

A. Separation of Microbes into Groups:

(i) Gram-Staining

A Danish scholar Christian Gram in 1884 devised a differential

staining procedure which differentiates bacteria into gram-positive and
gram-negative. This procedure is called Gram- staining technique.
This technique has experienced numerous modifications from time to
time and proves to be valuable for staining smears of pure cultures of
bacteria.

I. A thin film of young culture (smear) is fixed on a clean slide.

II. The smear is stained for one minute with ammonium oxalate crystal
violet. This stain sometimes yields over stained preparations in which
certain gram-negative organisms (e.g. Gonococcus) also retain the
stain. If this trouble is encountered, lesser amount of crystal violet
should be used.

III. The slide is washed in tap water for not more than 2 seconds to
remove excess stain.

IV. The slide is immersed for one minute in Lugol’s iodine solution.
The bacteria become deeply stained and appear deep purple in colour
due to crystal violet-iodine-complex formation.

V. The slide is washed in tap water and blot-dried.

49
 Fig. 4.2 Grams staining procedure

VI. The slide is gently agitated for 30 seconds in 95% ethyl alcohol
and blot-dried, gram-negative bacteria lose their stain in this step (i.e.
decolourize). However, the gram- positive ones retain deep purple
colour.

VII. The slide is now counterstained for 10 seconds in the safranin

solution.

VIII. The slide is washed in tap water, dried and examined.

Gram-positive bacteria: deep purple (blue); gram-negative bacteria:

pink (red).

50
Mechanism

Although different explanations have been given to answer why

bacteria respond differently to the Gram-stain, it seems likely that the
answer is related to the physical nature of their cell walls as when cell
wall is removed from gram-positive bacteria, they become gram-
negative. The peptidoglycan appears to act as a permeability barrier
preventing loss of crystal violet-iodine-complex. When gram-positive
bacteria are treated with destaining agent (alcohol), the alcohol is
thought to dehydrate the thick layer of peptidoglycan resulting in
shrinkage of pores of peptidoglycan. Shrinkage of peptidoglycan pores
prevents crystal violet-iodine-complex from escaping and the bacteria
remain deep purple.

In contrast, peptidoglycan is very thin in gram-negative bacteria, not

as highly crossed-linked as is in gram-positive ones, and has larger
pores. Alcohol, therefore, readily penetrates the lipid-rich outer layer of
the cell wall and extracts enough lipid thus increasing the porosity
further. For these reasons, alcohol more readily removes the deep
purple crystal violet-complex from gram-negative bacteria and the
latter become decolourized.

(ii) Acid-Fast Staining

The acid-fast stain is a differential stain developed first by Paul

Ehrlich in 1882 and later on modified by Ziehl- Neelsen, and is in use
even today by microbiologists. Majority of the bacteria are stained with
simple stain and Gram-stain but certain bacteria do not do so because
they have waxy components of the cell wall, hence their cell wall has
limited permeability. Such bacteria belong to genera like
Mycobacterium and Nocardia; and are stained by acid-fast stain; the

51
latter is used to identify Mycobacterium tuberculosis and M. leprae,
the pathogens responsible for tuberculosis and laprosy, respectively.
The acid-fastness property of these bacteria is correlated with high
lipid contents, which makes them difficult to stain. Hence for staining
of these bacteria heating with strong dye is required. Once the acid-
fast bacteria are stained, it is difficult to decolourize them even with
acid and alcohol. Moreover, acid-fast staining serves also as good
identification tool for a number of harmless saprophytic bacteria.

Carbol Fuchsin Stain

Basic fuchsin – 0.3 g

Ethanol (95%) – 10.0 ml

Phenol (heat melted crystals) – 5.0 ml

The basic fuchsin is dissolved in ethanol and phenol is dissolved in

water.

These two are mixed and kept for several days before filter and
use:

Decolourising solvent (acid-alcohol):

Ethanol (95%) – 97.0 ml

Hydrochloric acid (conc.) – 3.0 ml

Counter stain:

Methylene blue chloride – 0.3 g

Distilled water – 100.0 ml

Procedure:

The staining procedure is as follows:

52
I. A thin film of young culture (smear) is heat-fixed and air-dried on a
clean slide.

II. The smear is now flooded with carbol fuchsin.

III. The slide is steamed over boiling water for 3-5 minutes More stain
is added time to time to prevent smear from becoming dry.

IV. Slide is cooled and washed with distilled water until no colour
appears from the smear.

V. Smear is decolourized with decolourising solvent (acid-alcohol) for

15-20 seconds. Some bacterial cells appear red (faint pink) in colour,
while others decolourize. Slides arc washed with distilled water.

VI. Smears are now counterstained with methylene blue for 1-2
minutes and washed with distilled water.

VII. Slides are blot-dried with bibulous paper and examined directly
under oil-immersion.

Acid-fast bacteria appear red.

Non-acid-fast bacteria appear blue.

Mechanism:

Acid-fast staining helps classifying bacteria into two groups: acid-fast

and non-acid-fast. Acid fast bacteria, particularly those in the genus
Mycobacterium, do not bind simple stains but when stained by heating
with a mixture of carbol fuchsin (basic fuchsin + phenol) they retain
carbol fuchsin (the primary stain) after washing even with strong acid.
Once basic fuchsin penetrates with the aid of heat and phenol, cells of
acid-fast bacteria are not easily decolourized by an aid-alcohol wash
and hence remain red. This is due to the quite high lipid content of cell
walls of acid-fast bacteria; in particular, mycolic acid (a group of

53
branched chain hydroxy lipids) appears responsible for acid-fastness.
The nonacid-fast bacteria get decolourized after washing with acid-

Fig. 4.3 Acid Fast Stain

alcohol; they retain the counterstain methylene blue hence

appear blue.

Precautions

1. If necessary, more carbol fuchsin stain should be added to avoid its

evaporation and dryness.

2. Carbol fuchsin stain should be prevented from heating to avoid

“messy” preparations.

3. Over decolourization of the smear should be avoided.

54
B. Visualization of Various Structures

(i) Endospore Staining

Bacteria in the genera Bacillus and Clostridium form an

exceptionally resistant structure capable of surviving for long periods
in an unfavourable environment. This dormant structure is called an
endospore since it develops within the cell. Endospore morphology
and location vary with species and often are valuable in identification.
Endospores are not easily stained well by most dyes. Considerable
amount of heating is required in order to make the stain penetrate the
spore-coat, a thick wall primarily responsible for endospore resistance.
But once stained, they strongly resist decolourization. This property is
the basis of endospore staining techniques. However, there are two
staining procedures used by microbiologists to stain the endospores.
These methods are the Schaeffer-Fulton method and Dorner method.
For convenience, Schaeffer-Fulton method is given here.

The staining procedure of endospore by Schaeffer-Fulton

method is as follows

i. A thin film of young culture (smear) of endosporous bacteria is fixed

on a clean slide.

ii. The smear is heat-fixed on to the slide by gentle warming.

iii. Smear is covered with the solution of malachite green which is a

very strong stain that can penetrate the spore-coat of an endospore.

iv. The slide is kept on a suitable stand and heated with steam from
below for 5 minutes. If the stain dries up during heating, more stain is
added to the smear from time to time as per requirement.

v. The slide is washed gently under tap water.

vi. The slide is counter-stained with safrain for about 30 seconds.

55
vii. It is then washed with distilled water and dried with blotter.

viii. The smear is observed under oil immersion.

Fig. 4.4 Endospore staining

The endospore appears green while rest of the cell appears red.

Mechanism

Endospores are extremely resistant due to their thick wall, the

spore coat. The spore coat does not take the stain easily. Malachite
green, however, penetrates the spore coat of endospore after
considerable heating. Once stained, the endospore does not
decolourizes easily hence appears green even after washing. In
contrast, the counter stain fails entering the endospore but stains rest
of the cell content that appears red.

LET US SUM UP
The differentiation of bacteria into either the gram-positive or the
gram-negative group is fundamental to most bacterial identification systems.
This task is usually accomplished through the use of Gram's staining
method. Incidentally, the Gram stain is a deceptively simple procedure.
Staining can be performed quickly and easily but preparation and
interpretation of the smear requires considerable experience and training

56
and can therefore be prone to errors. The use of SOPs and Quality controls
are important in the performance of the test.

CHECK YOUR PROGRESS

1. Bacteria like Clostridium produced resistant structure called ------------

2. In negative staining the stain is ------------

GLOSSARY
1. Endospore - Bacteria in the genera Bacillus and Clostridium form an
exceptionally resistant structure capable of surviving for long periods
in an unfavourable environment. This dormant structure is called an
endospore since it develops within the cell.

SUGGESTED READINGS

1. Microbiology: Principles and Exploration by J. G. Black

ANSWERS TO CHECK YOUR PROGRESS

1. Endospores

2. anionic

MODEL QUESTIONS
1. Write about Bacterial staining

57
UNIT - 5

BACTERIAL REPRODUCTION
STRUCTURE

Overview

Learning Objectives
5.1 Binary Fission
5.2 Conidia
5.3 Budding
5.4 Cyst
5.5 Endospores
Let us Sum up
Check your Progress
Glossary
Suggested Readings
Answers to check your progress
Model questions

OVERVIEW
Bacteria, being single-celled prokaryotic organisms, do not have a
male or female version. Bacteria reproduce asexually.
In asexual reproduction, the "parent" produces a genetically identical copy
of itself.

LEARNING OBJECTIVES
To understand the Bacterial reproduction elaborately

5.1. BINARY FISSION

In binary fission, single cell divides into two equal cells. Initially the
bacterial cell reaches a critical mass in its structure and cellular constituents.
The circular double stranded DNA of bacteria undergoes replication, where

58
both the strands separate and new complementary strands are formed on
the original strands — results in the formation of two identical double
stranded DNA.

Fig. 5.1 Binary fission

The new double stranded DNA molecule i.e., incipient nuclei, are then
distributed into two poles of the dividing cell (no spindle formation takes
place like mitotic division). A transverse septum develops in the middle
region of the cell, which separates the two daughter cells. The binary fission
is a rapid process and cell undergoes division at an interval of 20-30
minutes. The division becomes gradually slow after certain time due to
accumulation of toxic substance and exhaustion of nutrients.

59
5.2. Conidia:

Conidia formation takes place in filamentous bacteria like

Streptomyces etc., by the formation of a transverse septum at the apex of
the filament. The part of this filament which bears conidia is called
conidiophore. After detachment from the mother and getting contact with
suitable substratum, the conidium germinates and gives rise to new
mycelium.

Fig. 5.2 Asexual reproduction A-Conidia and B-Budding

5. 3. Budding

The bacterial cell develops small swelling at one side which gradually
increases in size. Simultaneously the nucleus undergoes division, where one
remains with the mother and other one with some cytoplasm goes to the
swelling. This outgrowth is the bud, which gets separated from the mother by
partition wall, e.g., Hyphomicrobium vulgare, Rhodomicrobium vannielia, etc.

5. 4. Cysts

Cysts are formed by the deposition of additional layer around the

mother wall. These are the resting structure and during favourable condition
they again behave as the mother, e.g., many members of Azotobacter.

60
5.5. Endospore

Spores are formed during unfavourable environmental condition like

desiccation and starvation. As the spores are formed within the cell, they are
called endospores. Only one spore is formed in a bacterial cell. On
germination, it gives rise to a bacterial cell.

Endospore forming bacteria

1. Gram-positive

(a) Bacilli

(i) Obligate aerobes, e.g., Bacillus subtilis, B. anthracis.

(ii) Obligate anaerobes, e.g., Clostridium tetani, C. botulinum.

(b) Cocci, e.g., Sporosarcina.

2. Gram-negative

(i) Bacillus, e.g., Coxiella burnetii

(ii) Cocci, e.g., Escherichia coli

Some non-sporing anaerobic bacteria

1. Gram-positive

(a) Bacilli

(i) Lactobacillus

(ii) Propionibacterium

(iii) Bifidobacterium

(b) Cocci

(i) Peptococcus

(ii) Sarcina

(iii) Peptostreptococcus

2. Gram-negative

(a) Bacilli

61
(i) Fusobacterium

(ii) Leptotrichia

(iii) Bacteroides

(b) Cocci

(i) Acidoaminococcus

(ii) Veillonella

Shape and position of endospore:

Spores may be oval or spherical in shape. The position, relative size and
shape remain constant in a particular species. The position of spore may be
central, subterminal or terminal. In diameter, it may be the same or wider
(Clostridium) or less (Bacillus) than the width of the specific bacterial cell.

Fig. 5.3 Position of Endospore in Bacterial cell

Structure:
Endospore consists of a central protoplast, the core. The core is
mainly composed of DNA, ribosome, t-RNA, enzymes etc. The core is
covered by a thin membrane, called core membrane or inner membrane or
germ cell membrane, from which the cell wall of future vegetative bacterium
develops. It is covered by a thick layer, the cortex and then a multilayered
thin and tough outer spore coat, which may be differentiated into outer and
inner coat layer. In some species (Bacillus thuringiensis), it is covered by an
additional covering, called exosporium or exosporium basal layer, which is
apparently loose.

62
 Fig. 5.4 Endospore

Formation of endospore:

The endospore formation does not take place during active

phase of growth. The sporulation starts in conditions unfavourable for
the growth due to starvation, desiccation, high temperature etc. The
sporulation can also be induced by depleting S, C, N, Fe and
PO4 from culture medium. During sporulation, the first detectable
change is the conversion of compact nucleoid into an axial chromatin
filament. Then a transverse septum is laid down towards one pole,
which separates into small and large portion. The small portion with its
cytoplasm and DNA forms the fore spore, which later on develops into
a spore.

The membrane of large portion gradually grows around the fore

spore. The fore spore increases in size, which becomes opaque and
highly refractive, called the endospore. The entire process of
sporulation takes place within 16 to 20 hours. The cell in which spore is
formed, called sporangium, which remains viable for short period of time
after maturation of spore. The spore is liberated by autolysis of sporangium.
The spores can survive in different adverse conditions like heat, drying,
freezing, toxic chemicals and radiations. Some bacilli can resist a
temperature higher than 150°C.

63
The endospores germinate under favourable condition which consists
of three stages

(i) Activation

It takes place by the induction of one or more factors such as acidic

pH, heat (60°C for 1 hour), compounds containing free SH groups or through
abrasion.

(ii) Initiation

Binding of any effector substance like L-alanine, adenosine etc. of

the medium with the spore coat activates to form an autolysin. The autolysin
destroys the peptidoglycan of the cortex. Thereafter, water is taken up and
calcium dipicolonate is released. Dipicolonic acid helps to stabilise the spore
protein, and both dipicolonic acid and Ca ions provide resistance to heat.

(iii) Outgrowth

The swelling of spore wall and disintegration of the cortex help to

emerge a germ cell after breaking the spore coat, which behaves like a
vegetative cell. Each cell forms only one endospore and persists during
unfavourable condition. During favourable condition, it germinates and gives
rise to a single bacterial cell. So the spore is a perennating organ and the
process is called perennation rather than multiplication.

Fig.5.5 Bacterial growth curve

1. Lag Phase

64
 After inoculation into the sterile nutrient medium, the bacterium first
undergoes a period of acclimatisation. At that time, necessary enzymes and
intermediate metabolites are synthesised, thereby bacterium reaches a
critical stage before multiplication, multiplication takes place at this stage.
The duration of lag phase depends on the type of bacteria, quality of culture
medium, size of inoculum and several environmental factors such as CO2,
temperature, pH, etc. The average time of lag phase is 2 hours, although it
varies from species to species (1-4 hours).

2. Log Phase or Exponential Phase

In this phase, the bacteria undergo cell division and their population
(number) increase exponentially at a logarithmic rate. The number of viable
count, when plotted against time, gives a straight line of inclined fashion. The
average time of log phase is 8 hours, though it varies in different species.

3. Stationary Phase

In this phase, the growth i.e., cell division, almost ceases due to
exhaustion of nutrients and also the accumulation of toxic products. At this
stage the cell death starts at a slow rate and is compensated by the
formation of new cell through cell division. The total cell number increases at
a slow rate, but the viable count remains almost constant. The duration of
this phase is variable which ranges from few days to few hours. Secondary
metabolites like antibiotics, toxins etc. are produced in this phase.

4. Decline Phase

In the phase of decline, the total number of cells remains constant,

but the number of viable cells gradually decreases due to exhaustion of
nutrients and also the accumulation of toxic products. In some cases a few-
cells remain viable for long time, even after death of most of the cells. These
viable cells probably grow by utilising nutrients released from dead cells. The
cells attain maximum size at the end of lag phase and become smaller in log
phase (exponential phase). In spore forming species, the sporulation occurs
at the end of log phase (exponential phase) or in the early part of stationary
phase.

65
LET US SUM UP
Microbiology has had numerous significant applications for
human welfare. But, what would be the most promising areas for
future microbiological research. In comparison to other disciplines of
science, the mission of microbiology is clearer, Microbiology is
confident to its value due to its tremendous practical significance.
Future studies in the field of microbiology may lead to a better
understanding of the interactions between microorganisms and the
inanimate world. Among other things, this understanding should
enable us to more effectively control pollution. The microbiology of
tomorrow has to solve a variety of fundamental questions in biology.
For convenience, how do complex cellular structures develop and how
do cells communicate with one another and respond to the
environment.

CHECK YOUR PROGRESS

1. Conidia formation takes place in --------------------

2. Cysts are formed by the deposition of additional layer around --

-----------------------

GLOSSARY
1. Budding - The bacterial cell develops small swelling at one side
which gradually increases in size. Simultaneously the nucleus
undergoes division, where one remains with the mother and other
one with some cytoplasm goes to the swelling.

SUGGESTED READINGS

1. A Text Book of Microbiology by Maheshwari

ANSWERS TO CHECK YOUR PROGRESS

1. filamentous bacteria
2. the mother wall

66
MODEL QUESTIONS
1. Write about Endospores

67
 Block II

APPLIED MICROBIOLOGY

Unit 6: Spoilage of Food

Unit 7: Microbes in Fermented Products
Unit 8: Microbes in sewage treatment and Agriculture
Unit 9: Industrial Application of Microbes

68
UNIT - 6

SPOILAGE OF FOOD
STRUCTURE

Overview

Learning Objectives
6.1 Food Spoilage
6.2 Microbes in Household Products
6.3 Meat Spoilage
6.4 Spoilage of Vegetables and Fruits
6.5 Spoilage of Eggs
6.6 Cereal and Bakery Goods
6.7 Spoilage of Dairy Products
Let us Sum up
Check your Progress
Glossary
Suggested Readings
Answers to check your progress
Model questions

OVERVIEW
This unit deals with food spoilage. Spoilage of food can be defined as
any visible or invisible change which can makes food or product derived from
food unacceptable for human consumption. Spoilage not only leads to loss
of nutrients from food but also cause change in original flavor and
texture. Many microbes involved in the food spoilage. In this unit we can see
the microbes responsible for food spoilage.

69
LEARNING OBJECTIVES
To understand the spoilage in food by the microbes.

6.1 FOOD SPOILAGE

According to the Oxford English Dictionary to spoil is to ‘deprive

of good or effective qualities’. When a food is spoiled its
characteristics are changed so that it is no longer acceptable. Such
changes may not always be microbiological in origin; a product may
become unacceptable as a result of insect damage, drying out,
discolouration, staling or rancidity for instance, but by and large most
food spoilage is a result of microbial activity. Spoilage is also a
subjective quality; what is spoiled for one person may be perfectly
acceptable to another. The perception of spoilage is subject to a
number of influences, for example foods acceptable in some cultures
are unacceptable in others. Some products such as well matured
cheeses and game birds that have been hung for extended periods
are esteemed by some and highly objectionable to others.

Affluence is another contributory factor, many are not in the

position to be able to discard food due to some slight sensory defect,
in the 18th and 19th Century navy, sailors often preferred to eat in
dark corners so that they could not see the weevils and maggots in
their food. A general feature of microbial spoilage is its relatively
sudden onset – it does not appear to develop gradually, day by day a
little worse, but more often as an unexpected and unpleasant
revelation. This is a reflection of the exponential nature of microbial
growth and its consequence that microbial metabolism can also
proceed at an exponentially increasing rate. If a microbial product
associated with spoilage, for example an off odour, has a certain
detection threshold, the level will be well below this threshold for most
of the product’s acceptable shelf-life. Once reached however, it will be

70
rapidly passed so that a comparatively short time after, levels will be
well in excess of the threshold and the product will be profoundly
spoiled.

Fig. 6.1 Microbial growth and spoilage

Factors affecting food

Storage Conditions

Temperature and oxygen are considered two most important

factors that invite microbial contamination resulting in spoilage of
foods in storage conditions.

1. Role of temperature

Foods stored at below -17°C remain free from microbial growth

and a slow decrease in their population may even take place. Above
this temperature, the presence and multiplication of microorganisms in
food is usually recorded. This is the reason why refrigerated foods are
subject to spoilage by microorganisms. Food and food items stored at

71
room temperature or in warm conditions remain open for spoilage by
mesophilic and thermophilic microorganisms.

2. Role of oxygen:

Aerobic and anaerobic conditions play an important role in

determining the kinds of microorganisms which can multiply and spoil
various food and food-items in storage conditions. If oxygen is
available, various aerobic bacteria and moulds cause spoilage chiefly
surface spoilage, whereas if the conditions are anaerobic the spoilage
is caused by anaerobic, bacteria like Clostridium spp., etc.

2. Food’s Own Chemical Properties

The chemical conditions of foods influence the type of

microorganisms which can grow over and within it and hence
determine the nature of changes that would be brought by the
spoilage action of contaminating microorganisms. Four major
chemical conditions of food, e.g., composition, acidity, moisture, and
osmotic concentration are of major importance in this type of spoilage.

1. Chemical composition

(i) Foods rich in proteins are degraded by proteolytic microorganisms.

Proteins are degraded into its various components due to the action of
especially gram-negative, spore forming bacteria, e.g., Proteus,
Pseudomonas, some cocci, etc.

Protein foods + Proteolytic microbes → Amino acids + Amines +

Ammonia + Hydrogen sulfide

(ii) Foods rich in carbohydrates are degraded by carbohydrate

fermenting microorganisms, particularly yeasts and moulds. Bacteria
like Micrococcus, Leuconostoc, and Streptococcus can also degrade
carbohydrates.

72
Carbohydrate foods + Carbohydrate fermenting microorganisms →
Acids + Alcohols + Gases

(iii) Foods rich in fats are attacked by relatively few microorganisms

such as moulds and some gram- negative bacteria. These
microorganisms are, therefore, lipolytic in nature.

Fatty foods + Lipolytic microorganisms →Fatty acids + Glycerol

2. Acidity

Generally the fruits are acid foods (pH below 4.5) while nearly
all vegetables, fish, meats, and milk-products are non-acid (pH above
4.5). Since the pH of the acid foods (fruits) is sufficiently low, they do
not allow bacterial growth and subsequent spoilage. They are spoiled
mainly by yeasts and moulds. Contrary to this, non-acid foods have
sufficiently high pH and are spoiled mainly by bacteria.

3. Moisture and osmotic concentration

Average 13% free water is required in food for usual microbial

growth. This is the reason why the foods of high sugar and salt
concentrations do not allow most of the microorganisms to grow. But,
specific microbial growths cannot be over-ruled. 65-70% sugar
concentration is required to prevent mould-growth and 50% to prevent
bacterial and yeast growth.

6.3 MICROBES IN HOUSEHOLD PRODUCTS

Microbes and their products are used in everyday life like

production of curd, formation of dough, cheese, etc.

Production of Curd

Microorganisms like Lactobacillus and other Lactic Acid

Bacteria (LAB) grow in milk, which convert it into curd. We have seen
at home that a starter is added to milk which turn it into curd. This

73
starter is known as inoculum, which contains millions of bacteria.
During growth, bacteria produce acids that coagulate and partially
digest the milk proteins. Thus, converting milk into curd. These also
improve nutritional quality by increasing vitamin-B12 content of the
curd. Lactic acid bacteria also play very beneficial role in checking
disease causing microbes in our stomach.

Fermentation by Microbes

(i) Dough

It is fermented by bacteria in making foods such as dosa and

idli. The puffed up appearance of dough is due to the production of
CO2 during fermentation. In bread making, dough is fermented using
baker’s yeast, i.e., Saccharomyces cerevisiae.

(ii) Toddy

It is a traditional drink of some parts of Southern India. It is

made by yeast fermentation sap from palms trees, coconut, etc.
Microbes are also used to ferment fish, soya bean, bamboo shoots,
etc.

(iii) Cheese

It is known to be the oldest food item in which microbes are

used. It is formed by partial degradation of milk by different
microorganisms. Different varieties of cheese are known by their
texture, flavour and taste. Swiss cheese with large holes is produced
by Propionibacterium sharmanii. Holes are created due to the
production of large amount of CO2 produced by this bacterium.
Roquefort cheese is ripened by growing a specific fungi on them,
which give them a particular flavour.

74
6.3 MEAT SPOILAGE

1. Processed meats (hot dogs, sausage and luncheon meats)

These products are composed of a variety of blended

ingredients, any of which can contribute microorganisms to the food.
Yeasts and bacteria are the most common causes of spoilage, which
is usually manifest in 3 ways:

Slimy spoilage

Like other meat products, this occurs on the surface and is

caused by the buildup of cells of yeasts, lactobacilli, enterococci
or Brochothrix thermosphacta. Washing the slime off with hot water
can restore the product quality.

Sour spoilage

From growth of lactic acid bacteria (which originate from

contaminated ingredients like milk solids) under the casing. These
organisms ferment lactose and other CHOs in the product and
produce organic acids. Taste is adversely affected but the product is
not harmful if eaten. Greening due to H2O2 or H2S production.
Because greening indicates more extensive product breakdown, I
would not recommend eating green wieners. Reasons Cured meats
(bacon, hams) are resistant to spoilage:

1. Use of nitrite/nitrate

2. Smoking or brining of hams

3. The high fat content (thus low aw) of bacon

Instead, spoilage of these products is often caused by molds from

several genera including Aspergillus, Fusarium, Mucor, Penicillium,
Rhizopus and Botrytis.

2. Poultry

75
 a. general trends are the same as other fresh meats

b. similar microflora on fresh birds

c. whole birds have lower counts than cut-up parts

d. additional processing steps add to the microbial load

When poultry is in the advanced stages of spoilage, the skin will often
fluoresce under UV because so many fluorescent pseudomonads are
present. Off odors generally appear before sliminess develops. The
same bacteria can produce visceral taint, a condition manifest by off
odors in the abdominal cavity or poultry.

3. Fish

a. Fish have high nitrogen content but no carbohydrate.

b. The microbial quality of fish and especially shellfish is

heavily influenced by the quality of the water from which they were
harvested.

Unsanitized processing steps are principal culprits in fish

products with high microbial loads. In general, frozen fish products
have lower counts than fresh products. Bacteria on fresh fish are
concentrated on the outer slime, gills and intestine. Spoilage of salt-
and freshwater fish occurs in similar ways; the most susceptible part
of the fish to spoilage is the gill region, and the best way to detect
spoilage in fresh fish is to sniff this area for off odors produced
by Pseudomonas and Acinetobacter-Moraxella bacteria. The odors
include ammonia, triethylamine, H2S and other compounds. If fish are
not eviscerated quickly, bacteria will move through the intestinal walls
and invade the meat that lies next to the abdominal cavity. Spoilage
of crustaceans (shrimp, lobsters, crabs and crayfish) is similar, but
these products have some CHO (0.5%) and more free amino acids so
spoilage can occur more rapidly.

76
Mollusks (oysters, clams, mussels, squid and scallops) have more
CHO (3-5%) and less nitrogen than either fish or shellfish. Microflora
of mollusks can vary a great deal depending on the quality of the
water from which they were harvested. Shellfish are filter feeders and
can be expected to contain almost any microorganism or virus that
occurs in the water where they were obtained. If these products were
taken from clean waters, then the
usual Pseudomonas and Acinetobacter-Moraxella types of spoilage
bacteria dominate.

6.4 SPOILAGE OF VEGETABLES AND FRUITS

Vegetables are a good substrate for yeasts, molds or bacteria It

is estimated that 20% of all harvested fruits and vegetables for
humans are lost to spoilage by these microorganisms. Because
bacteria grow more rapidly, they usually out-compete fungi for readily
available substrates in vegetables. As a result, bacteria are of greater
consequence in the spoilage of vegetables with intrinsic properties
that support bacterial growth (favorable pH, Eh). One of the most
common types of bacterial spoilage caused by Erwinia carotovora and
sometimes by Pseudomonas spp., which grow at 4oC Softening can
also be caused by endogenous enzymes.

In vegetables where bacterial growth is not favored (e.g. low

pH), molds are the principal spoilage agents. Most molds must invade
plant tissue through a surface wound such as a bruise or crack.
Spores are frequently deposited at these sites by insects
like Drosophila melanogaster, the common fruit fly. Other molds
like Botrytis cinerea, which causes grey mole rot on a variety of
vegetables, are able to penetrate fruit or vegetable skin on their own.

Fruits

77
 Like vegetables, fruits are nutrient rich substrates but the pH of
fruits does not favor bacterial growth. As a result, yeasts and molds
are more important than bacteria in the spoilage of fruits. Several
genera of yeasts can be found on fruit. Because these organisms
grow faster than molds, yeast often initiate fruit spoilage. Then molds
finish the job by degrading complex polysaccharides in cell walls and
rinds.

Specific Spoilage Organisms:

1. Blue rot – Penicillium, fruits

2. Downy mildews – Phytophora, large masses of mycellium (grapes)

3. Black rot – Aspergillus, onions

4. Sour rot – Geotrichum candidum

6.5 SPOILAGE OF EGGS

Eggs have several intrinsic parameters which help to protect the

nutrient-rich yolk from microbial attack. These include the shell and
associated membranes, as well as lysozyme, conalbumin, and a high
pH (>9.0) in the white. Freshly laid eggs are generally sterile, but
soon become contaminated with numerous genera of bacteria.
Eventually, these MO will penetrate the eggshell and spoilage will
occur. Pseudomonads are common spoilage agents, but molds
like Penicillium and Cladosporium sometimes grow in the air sac and
spoil the egg.

6.6 CEREAL AND BAKERY GOODS

These products are characterized by a low aw which, when stored

properly under low humidity, restricts all micro organisms except
molds. Rhizopus stolonifer is the common bread mold, and other
species from this genus spoil cereals and other baked goods. -

78
Refrigerated frozen dough products have more water and can be
spoiled by lactic acid bacteria.

6.7 SPOILAGE OF DAIRY PRODUCTS

Milk

Milk is a very rich medium. Raw milk may contain many

microorganisms found on the cow hide (which incl. soil and fecal
bacteria), udder, and milking utensils. It can also include G -ve, G+ve
yeasts and molds. When properly handled and stored, the flora of
pasteurized milk is primarily G+ bacteria. Psychrotropic
pseudomonads are common in bulk stored raw milk -produce heat
stable enzymes that can reduce milk quality and shelf life.
Pasteurization kills most G- (incl. Pseudo.), yeasts and molds -some
G- enzymes, thermotolerant G+ bacteria and spores survive -
Psychrotropic Bacillus spp. are also common in raw milk

Pasteurized fluid milk – spoiled by a variety of bacteria, yeasts and

molds. In the past, milk was usually soured by LAB such as
enterococci, lactococci, or lactobacilli, which dropped the pH to 4.5
where milk proteins coagulate (curdling). Today, milk is more
frequently spoiled by aerobic sporeformers such as Bacillus, whose
proteolytic enzymes cause curdling. Molds may grow on the surface of
spoiled milk, but the product is usually discarded before this occurs.

Butter; high lipid content and low aw make it more susceptible to

surface mold growth than to bacterial spoilage. Some pseudomonads
can be a problem; “surface taint” -putrid smell, caused by the
production of organic acids (esp. isovaleric) from P. putrefaciens
Rancidity due to butterfat lypolysis caused by P. fragi are common.

Cottage cheese can be spoiled by yeasts, molds and bacteria. The

most common bacterial spoilage is “slimy curd” caused

79
by Alcaligenes spp. (G- aerobic rod bound in soil, water, and intestinal
tract of vertebrates). Like Campylobacter, these species do not oxidize
CHOs but instead use amino acids and TCA intermediates.
Penicillum, Mucor and other fungi also grow well on cottage cheese
and impart stale or yeasty flavors.

Ripened Cheeses – (1) low aw, (2) low pH and (3) high salt inhibit
most spoilage microorganisms except surface mold growth. Spores
of C. butyricum, C. sporogenes and others can germinate in cheeses
(e.g. Swiss) with intrinsic properties that are less inhibitory (e.g. lower
salt, higher pH). -These organisms may metabolize citrate, lactose,
pyruvate or lactic acid and produce butyrate or acetate plus CO 2 or
H2 gas which “blows” the cheese.

LET US SUM UP
Spoilage of Food predominantly carried out by microbes. Microbes
can easily spoil cooked food items very quickly and fresh fruits an d
vegetables also spoil.

CHECK YOUR PROGRESS

1. Curd spoiled by -----------------

2. Onions spoiled by -----------------------

GLOSSARY

1. Slimy curd – bacterial spoilage of cottage cheese

SUGGESTED READINGS

1. Food Microbiology by M. R. Adams

ANSWERS TO CHECK YOUR PROGRESS

80
 1. Lactobacillus

2. Aspergillus

MODEL QUESTIONS
1. Write detailed account on Food spoilage.

81
UNIT - 7

MICROBES IN FERMENTED
PRODUCTS

STRUCTURE

Overview

Learning Objectives
7.1 Fermented Beverages
7.2 Antibiotics
Let us Sum up
Check your Progress
Glossary
Suggested Readings
Answers to check your progress
Model questions

OVERVIEW
Microbes are used to synthesizes a number of products
valuable to human beings in industries also e.g., beverages and
antibiotic. For industrial production, microbes are grown in very large
vessels called fermenters.

LEARNING OBJECTIVES
To know the products of enzymes and beverages are produced
from the fermentation by microbes..

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7.1 FERMENTED BEVERAGES

Yeasts have been used from ancient time for the production of
beverages like wine, beer, whisky, brandy and rum. Saccharomyces
cerevisiae commonly called brewer’s yeast is used for bread making,
fermenting malted cereals and fruit juices to ethanol. Depending upon
the type of raw material and processes, different types of alcoholic
drinks are prepared. Wine and beer are filtered, pasteurized and
bottled without further distillation, whereas whisky, brandy and rum are
produced by the distillation of fermented broth. Beer has an alcoholic
content of 3-6%, while in wines; the alcoholic content is around 9-
12%.

Wine production
Wine is made from grapes or other fruit. The grapes are first cleaned
of leaves and stems and the fruit is crushed into must that is ready for
fermentation. The yeasts used for the fermentation grow a film on the fruit or
in the environment. These wild strains play an important role in the final
properties of the drink. However, cultivated strains of Saccharomyces
cerevisiae are often added to improve the consistency of the final product.
There are hundreds of commercially available yeast strains for wine
fermentation.

Beer production
Beer is the most consumed alcoholic beverage in the world. It is
made most often of malted barley and malted wheat. Sometimes a mixture
of starch sources can be used, such as rice. Unmalted maize can be added
to the barley or wheat to lower cost. Potatoes, millet and other foods high in
starch are used in different places in the world as the primary carbohydrate
source.
Vinegar
Vinegar is a food product made by acetic acid bacteria that can
ferment the alcohol in alcoholic liquids to acetic acid.

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7.2. ANTIBIOTICS

The term ‘antibiotics’ was coined by Waksman (1942). The

name antibiotic is derived from the Greek words against and bios—
life, together they mean ‘against life’ (with reference to disease
causing organisms). These are the chemical substances, produced by
some microbes and can kill or retard the growth of other disease
causing microbes. The first antibiotic discovered was Penicillin.
Alexander Fleming, while working on Staphylococci bacteria, found a
chemical, which inhibits the bacterial growth. It was named as
penicillin after the mould Penicillium notatum. The potential use of
Penicillium as antibiotic was established by Ernest Chain and Howard
Florey. Penicillium was extensively used in treating American soldiers
wounded in World War II. Chain and Florey were awarded the Nobel
Prize in 1945, for this discovery. Some other antibiotics were also
purified after the successful discovery of Penicillium. They greatly
improved the capacity to treat deadly diseases like plaque, whooping
cough (kali khansi), diphtheria (gal ghotu), leprosy (kusht rog), which
use to kill millions of people all over the world, etc. As pathogens often
develop resistance to existing antibiotics, so, the newer antibiotics are
required to be produced. The good antibiotics should be harmless to
host with no side effects and should have the ability to destroy
pathogens. Antibiotics are mainly obtained from Actinomycetes,
eubacteria and fungi, etc.

Common antibiotics

1. Penicillin - Penicillium notatum

2. Streptomycin - Streptomyces griseus
3. Cephalosporins - Cephalosporium acremonium
4. Erythromycin - Streptomyces erythreus

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 5. Gentamicin - Micromonospora purpurea
6. Rifamycin - Streptomyces mediterranei

7.3. CHEMICALS, ENZYMES AND OTHER BIOACTIVE

MOLECULES

Microbes are being used for the commercial and industrial

production of certain chemicals like alcohols, organic acids and
enzymes. The other molecules, which are functional in living systems
or can interact with their components are called bioactive molecules.
Enzymes are very well established in biotechnology and the microbes
are also used in their production.

85
LET US SUM UP
The vast majority of novel antibiotics have been detected by
screening of wild organisms obtained from soil and other natural habitats.
Although a wide taxonomic range of microbes have the ability to produce
antibiotics.

CHECK YOUR PROGRESS

1. Microbe involves in wine production is -------------

2. Cephalosporin is a product of ----------------

GLOSSARY

1. Vinegar – Fermented alcoholic product

SUGGESTED READINGS

1. Industrial Microbiology by [Link]

ANSWERS TO CHECK YOUR PROGRESS

1. Yeast
2. Cephalosporium acremonium

MODEL QUESTIONS
1. Write about antibiotics and microbes used for the production of
antibiotics

86
UNIT 8

MICROBES IN SEWAGE TREATMENT

AND AGRICULTURE
STRUCTURE

Overview

Learning Objectives
8.1 Small scale sewage treatment
8.2 Large scale sewage treatment
8.3 Biological Oxygen Demand
8.4 Microbes in Production of Biogas
8.5 Microbes as Bio-control agents
8.6 Biological control of Pests and Diseases
8.7 Microbes as Biofertilizers
Let us Sum up
Check your Progress
Glossary
Suggested Readings
Answers to check your progress
Model questions

OVERVIEW
Sewage refers to the municipal wastewater generated every
day in cities and towns. Human excreta are a major component of it. It
contains large amount of organic matter, microbes and pathogens out
of which many are pathogenic. It cannot be discharged into natural
water-bodies like rivers, streams, etc., because it not only contains
human excreta and other organic wastes but a number of pathogenic
microorganisms too. Before disposal, sewage has to be treated in a

87
Sewage Treatment Plants (STPs) in order to make it less polluting.
The treatment of wastewater is done by the heterotrophic microbes,
naturally present in the sewage.

LEARNING OBJECTIVES
To understand role of microbes in Agriculture and sewage
treatments.

8.1 SMALL SCALE SEWAGE TREATMENT

There are several methods of sewage treatment on small scale.

A few of them are described below

(i) Cesspools

Human waste is thrown in cesspools in many homes. It is

constructed in underground part with concrete in such a way that it
contains wall of cylindrical rings with pores. Its opening is near the
ground level. Wastewater (sewage) enters the cesspool through the
inlet pipe. The bottom of cesspool remains open. Therefore, the
suspended solid material falls on the bottom of cesspool and forms
sludge after getting deposited in huge amount.

Fig. 8.1 Cesspool

88
 Water passes out through the open bottom of cesspool and
through pores into the surrounding soil. The organic materials of the
sludge are decomposed by anaerobic bacteria resulting in release and
deposition of breakdown products on the ground.

Thus the amount of breakdown products exceeds; it forms thick

layers which need to be cleaned by using strong acids. Dried bacterial
preparation of Bacillus subtilis or yeast cells should be added at
intervals. These accelerate the decomposition of sludge deposited at
the bottom of cesspool.

(ii) Septic Tanks

In rural areas, individual family uses septic tank because of lack

of public sewers. Septic tank is a metallic or concrete tank which is
kept below the ground level somewhere near the homes. Into septic
tanks all the domestic wastes flow through the inlet pipes. A family of
four members needs a septic tank of 3 × 5 × 5 feets by accumulating
about 750 gallons of sewage.

The suspended organic materials are accumulated at the

bottom of tank, whereas the water flows through outlets to a
distribution box which is connected with perforated pipes that open
under the soil surface in the surrounding areas. Therefore, effluents
from the tank are passed to underground surface of soil. Through this
process, the pathogenic microbes are not eliminated. Therefore, the
drinking water supply must be kept at certain distance of the pipes of
septic tanks.

89
 Fig. 8.2 Septic tank model

The organic materials accumulated in septic tank is

decomposed by anaerobic bacteria releasing into water several by-
products such as sugars, alcohols, organic acids, amino acids, fatty
acids, glycerols and gases (e.g. H2, H2S, CH4, CO2, etc.). However,
there remain undigested organic materials called sludge. Sludge is
removed from the septic tank at certain intervals by pumping process
otherwise it will block the tank and pipes. Sludge acts as a source of
humus when applied in field. In addition, in small towns sewage is
collected into large ponds which are called oxidation lagoons. The
sewage is discharged into oxidation lagoons where organic materials
are oxidized first by aerobic organisms and the sediments are
decomposed by anaerobic microorganisms.

90
8.2 LARGE SCALE SEWAGE TREATMENT

Sewage treatment on a large scale of populations of city is

known as large scale sewage treatment. In cities sewage and garbage
are generated in massive amount per day which is treated by
municipal plants. A schematic view of waste treatment by a municipal
plant is shown in. Overall processes of conventional municipal sewage
plant can be divided into three steps: the primary treatment,
secondary treatment, and tertiary treatment.

The primary treatment is viewed for physical separation of

insoluble materials, to lower BOD; the secondary separation is based
on microbial decomposition of organic materials in the effluents; the
tertiary treatment is the chemical removal of inorganic nutrients and
pathogenic microbes.

Fig. 8.3 Flow chart of Sewage treatment

91
Table 8.1 : Major steps in primary, secondary and tertiary
treatment of wastes.

(i) Primary Treatment

Primary treatment is the physical removal of 20-30% of organic

materials present in sewage in particulate form. The particulate
material is removed by screening, precipitation of small particulate and
settling in basin or tanks where the raw sewage is piped into huge and
open tanks. The solid material (sludge) is removed and kept in
landfill/composting for anaerobic digestion. The liquid portion is piped
into sludge tanks. The accumulated materials in sludge tanks are
subjected to aluminium sulfate or the other coagulants so that the
suspended particles, organic materials and microorganisms should be
trapped as in sedimentation process of water purification.

(ii) Secondary Treatment

Secondary treatment is also called biological treatment or

microbial degradation. By this process about 90-95% of the BOD and
many pathogens are removed. There are several means by which
BOD can be reduced in secondary treatment. Reduction of BOD by
90% is achieved through mineralization of small fraction of organic
matter and conversion of large proportion to removable solids. The
microbial activities may be aerobic or anaerobic.

92
Secondary treatment is done by several methods as described
below:

(a) The oxidation ponds

The oxidation ponds (also called lagoons or stabilization ponds)

permit the growth of algal forms on waste-water effluent. It is used for
secondary treatment in rural areas or industrial sectors.

The organic materials are degraded by heterotrophic bacteria

into simpler forms that in turn support the growth of algae. Algae use
these nutrients to increase their biomass. Air supplies oxygen for
biochemical oxidation of organics. Oxygen evolved by algae after
photosynthesis maintains the oxygen deficit created by heterotrophs.
The efficiency of oxidation process can be improved by constructing
shallow ponds.

Fig. 8.4 Oxidaion pond

The algae growing in oxidation ponds are: Chlorella pyrenoidosa, C.

ellipsoides, Scenedesmus acutus, S. quadricauda, Spirulina platensis,

93
etc. Secondary treatment through oxidation ponds is the aerobic
sewage treatment device.

(b) The trickling filter

Aerobic secondary treatment also can be carried out with a

trickling filter. It is a simple sewage treatment device that consists of a
bed of a crushed stone, gravel, slag, or synthetic material with drains
made at the bottom of the tank. Thus the trickling filter has a pile of
rocks over which sewage or organic wastes slowly trickle.

Fig. 28.5 Bacterial slime Pseudomonas

A revolving sprinkler (arm) is suspended over a bed of porous

material which distributes the liquid sewage over it, and collects the
effluents at the bottom. Due to spraying process, sewage is saturated
by oxygen.

The porous filter bed becomes coated with slimy bacterial

growth mainly by Zooglea ramigera and other slime producing
bacteria. The slime is colonised by the heterotrophic microorganisms
e.g. bacteria (Beggiatoa alba, Sphaerotilus natons, Achromobacter
spp., species of Pseudomonas and Flavobacterium), fungi,
nematodes, protozoa, etc. These microorganisms form a stationary
microbial culture because of continuous supply of nutrients present in
sewage and metabolising the organic constituents into the more stable

94
end products. Therefore, BOD of effluent is reduced by these
microorganisms. The microorganisms get air through porous bed. A
newly constructed bed needs a few weeks to function efficiently
unless the zoogleal film is coated over it.

Fig. 8.6 Trickling filter

(c) The activated sludge

It is also one of the widely used aerobic treatment systems, for

waste water in which very vigorous aeration of the sewage is done.
The sewage is passed into an aeration tank from primary settling tank.
Sewage is aerated by mechanical stirring. Due to vigorous aeration of
sewage floc-formation occurs. The colloidal and finely suspended
matter of sewage form aggregates which are called floccules. The
floes are permitted to settle down in secondary settling tank. The
particles of floe i.e. activated sludge contain large amount of
metabolising bacteria together with yeasts, fungi and protozoa. The
activated sludge is introduced in primary settling tank and aeration
tank just for rapid development of microorganisms and rapid

95
exploitation of organic matter. This process is repeated i.e. addition of
settled sludge to fresh sewage, aeration, sedimentation, addition of
settled sludge to fresh sewage, and so on. This repeating process
results in complete flocculation of fresh sewage within a few hours.
Activated sludge process reduces the BOD of effluent to 10-15% as
compared to raw sewage.

Fig.8.7 Aerobic activated sludge

The use of activated sludge hastens the efficiency of system. A

poor settlement of activated sludge floes adversely affects the
efficiency of sewage treatment plant. The microorganisms found in
activated sludge floes are the heterotrophs such as Gram-negative
rods (e.g. E. coli, Enterobacter, Pseudomonas, Achromobacter,
Flavobacterium, Zooglea, etc.), Arthrobacter, Corynebacterium,
Mycobacterium, Sphaerotilus, large filamentous bacteria, some
filamentous fungi, yeasts and protozoa. These microbes secrete slime
that holds floes.

Thus, the floes are microbial biomass held together by slime.

The settled sludge should be removed from settling tank time to time
otherwise poor settling will result in bulking of sludge. The bulking

96
sludge is caused by massive development of filamentous bacteria
(e.g. Sphaerotilus, Baggiatoa, Thiothrix), and filamentous fungi (e.g.
Cephalosporium, Cladosporium, Geotrichium, etc.). Thus, the settled
sludge is permitted to anaerobic treatment and reinoculation of fresh
sewage.

(d) Anaerobic digesters

All the aerobic processes produce excess microbial biomass or

sewage sludge which contains many recalcitrant organics. The sludge
from aerobic sewage treatment together with the materials settled
down in primary treatment is further treated in anaerobic digesters
through the process of anaerobic digestion. These digesters are used
only for processing of settled sewage sludge and the treatment of very
high BOD industrial effluents. Anaerobic digesters are large
fermentation tanks designed to operate anaerobically with continuous
supply of untreated sludge and removal of final, stabilized sludge
product. However, in these tanks provisions are made for mechanical
mixing, heating, gas collection, sludge addition and removal of final
stabilized sludge. High amount of suspended organic materials with
high number of bacterial community (109 – 1010 CFU/ml) is found. The
organics are decomposed by a number of anaerobic microorganisms
whose population is found to be 2 – 3 times greater than the
anaerobes.

Anaerobic digestion involves the following three steps

Fermentation

The fermentation of sludge components to form organic acids

(including acetate) from organic polymers is done by a number of
bacteria such as species of Bacteroides, Clostridium,
Peptostreptococcus, Eubacterium, Lactobacillus, etc. The organic

97
acids are butyrate, propionate, lactate, succinate, acetate along with
ethanol and H2, CO2, etc.

Fig. 8.8 Anaerobic Sludge digester

i. Acetogenic reactions

The products (e.g. butyrate, propionate, lactate, succinate,

ethanol) produced during fermentation are utilized as substrate by
several acetogenic bacteria viz., Syntrophomonas, Syntrophobacter
and Acetobacterium. The products produced as a result of acetogenic
reactions are: acetate, H2 and CO2.

ii. Methanogenesis

The products produced during acetogenesis are utilized as

substrate by methanogenic bacteria. Acetate is used to produce CH 4 +
CO2 by Methanosarcina and Methanothrix. H 2 and HCO3– are used to

98
produce methane by several bacteria e.g. Methanobrevibacter,
Methanomicrobium, Methanogenium, Methanobacterium,
Methanococcus, and Methanospirillum. A critical balance between
oxidants and reductants is maintained during methanogenic
processes. The hydrogen concentration must be maintained at a low
level so that it can function most efficiently. Upon accumulation of
hydrogen and organic acids, methane production is inhibited. Thus,
the final product of anaerobic digestion is a mixture of gases (70%
CH4, 30% CO2), microbial biomass and non-biodegradable residues
(e.g. heavy metals, polychlorinated biphenyls, etc.).

8.3 BIOCHEMICAL OXYGEN DEMAND (BOD)

BOD refers to the amount of the oxygen that would be

consumed if all the organic matter in one liter of water was oxidised by
bacteria.

(a) The greater the BOD, more polluting water will result. So, the
sewage water is treated till the BOD is reduced.

(b) When the BOD of effluent is reduced significantly, the effluent is

then passed into a settling tank, where the bacterial ‘floes’ are allowed
to sediment called activated sludge.

(c) A small part of the activated sludge is then pumped back into the
aeration tank to serve as the inoculum. Then the remaining part of the
sludge is pumped into large tanks called anaerobic sludge digesters,
in which other anaerobic bacteria are also present.

(d) They digest the organic mass as well as aerobic microbes bacteria
and fungi of the sludge. During the digestion, gases like methane,
hydrogen sulphide (H2S), carbon dioxide (CO2) etc., are produced.

(e) These gases form biogas that are used as a source of energy
because these are inflammable.

99
(f) The effluent from secondary treatment plant is released into natural
water-bodies like rivers and streams.

8.4 MICROBES IN PRODUCTION OF BIOGAS

Biogas is a mixture of gases, but the major content is methane

gas. It is produced by the microbial activity in digestion of biomass
with the help of certain bacteria. Biogas is used as fuel.

The type of gas produced depends upon the microbes and the
organic substrates they utilise. Certain bacteria, which grow
anaerobically on cellulosic material, produce large amount of methane
along with CO2and H2. These bacteria are called methanogens.
Methanogens produce large amount of methane (50-70%), CO2 (30-
40%) and H2. Methanogens, are also present in anaerobic sludge
during sewage treatment. They are also present in rumen (a part of
stomach) of cattle, where they help in breakdown of cellulosic material
in the food and thus, play important role in nutrition of cattle.

Biogas (Gobar Gas) Plant

These plants are mostly functional in rural areas, where dung

can be used for the generation of biogas. The excreta of cattle
commonly called gobar is rich in methanogenic bacteria. Cattle dung
is available in large quantities in rural areas hence; it is a good choice
for the production of biogas. Biogas plant consists of a concrete tank
(10-15 feet deep) in which bio-wastes are collected and slurry of dung
is fed. A floating cover is placed over the slurry, which keeps on rising
as the gas is produced in the tank. Methanobacterium in the dung acts
on the bio-waste to produce biogas. An outlet is also present which
connects to a pipe that supply biogas to the nearby house. There is an
another outlet from which spent slurry is being removed that can be
used as fertiliser.

100
 Fig. 8.9 Biogas plant

The biogas thus produced is used for cooking and lighting. Biogas fuel
technology was developed in India mainly by Khadi and Village
Industries Commission (KVIC) and Indian Agricultural Research

8.5 MICROBES AS BIO-CONTROL AGENTS

Bio-control is the use of biological methods for controlling plant

diseases and pests. These chemicals are also harmful for human
beings and animals. Thus, polluting the environment (soil,
groundwater).

(a) Chemical pesticides decrease the growth of weeds, reduce attack

from pathogens and drive away or kill insects, worms and birds, which
happen to feed on crop plants.

(b) These undesirable species can range from agricultural pests to

water contaminants to virulent pathogens. They are undesirable
because these species are a detriment to human interests in an
ecosystem.

(c) Microbes used for bio-control reduce the target species population
through many ecological mechanisms, including pathogenism,
competition, production of allelochemicals and other interactions.

101
(d) Bacteria, fungi and viruses can all act as bio-control agents due to
the large diversity of target species and the variety of methods of
action. The important examples of microbial bio-control agents include
Bacillus thuringiensis, Pseudomonas and Beauveria bassiana.

8.6 BIOLOGICAL CONTROL OF PESTS AND DISEASES

Bio-control is a holistic approach that seeks to develop an

understanding of the interactions between various organisms and use
this knowledge to control pests, weeds, etc. Bio-controlling requires
familiarity with various life forms, their habitat, predators, life style,
etc., to use them in bio-control measures and reducing the
dependence on chemicals and pesticides. Bio-control microbes
control their target species through a web of biological interactions.

Some examples of biological control agents are given as under

(a) Ladybird and dragonflies are useful to get rid of aphids and
mosquitoes respectively.

(b) To control butterfly, caterpillars, bacteria, such as. Bacillus

thuringiensis is used in the form of sprays.

(c) Using genetic engineering methods. Bacillus thuringiensis toxin

genes are introduced into plants. Such plants are resistant to attack by
insect pests. Bt cotton is one such example.

(d) Trichoderma species are free-living fungi that are very common in
the root ecosystems. They are effective bio-control agents of several
plant pathogens.

(e) Baculoviruses belonging to the genus Nucleo poly hedro virus are
viruses used in biological control. These are excellent for species-
specific, narrow spectrum insecticidal applications. They are used in
integrated pest management programme. They do not have any
negative impact on the ecosystem.

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8.7 MICROBES AS BIO-FERTILISERS

The chemical fertilisers are being used in increasing amounts in

order to increase the output in high yielding varieties of crops.
However, due to the excessive pollution because of these chemical
fertilisers, there is pressure to shift to organic farming i.e., to use
biofertilisers. Biofertilisers are the organisms that bring about nutrient
enrichment of soil by enhancing the availability of nutrients to the
crops.

The main sources of bio-fertilisers are as follows:

1. Bacteria

Rhizobium is a symbiotic bacterium that lives in the root

nodules of legumes and fixes atmospheric nitrogen into organic
compounds. Azotobacter and Azospirillum are free-living bacteria,
which absorb free nitrogen from the soil, air and convert it into salts of
nitrogen compounds like amino acids and enrich the soil nutrients.
Nitrogen-fixing bacteria fix atmospheric nitrogen into organic form,
which is used by the plant as nutrient.

2. Fungi

It also forms symbiotic association with plants called as

mycorrhiza, which absorb phosphorus from soil and pass it on to the
plants. Many members of genus Glomus form mycorrhiza.

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Plants with mycorrhizal association show other benefits also
such as

(i) Resistance to root borne pathogens.

(ii) Tolerance to salinity and drought.

(iii) Increase in plant growth and development.

3. Cyanobacteria

These are autotrophic microbes found in aquatic and terrestrial

environments. Many of them fix-nitrogen, e.g., Anabaena, Nostoc,
Oscillatoria, etc. In paddy fields, cyanobacteria serve as important bio-
fertilisers, so blue-green algae also add organic matter to the soil, thus
increasing the fertility.

Mycotoxins

Mycotoxins are a group of naturally occurring chemicals

produced by certain moulds. They can grow on a variety of different
crops and foodstuffs including cereals, nuts, spices, dried fruits,
apple juice and coffee, often under warm and humid conditions. The
mycotoxins of most concern from a food safety perspective include:

 aflatoxins (B1, B2, G1, G2 and M1)

 ochratoxin A

 patulin toxins produced by Fusarium moulds, including

fumonisins (B1, B2 and B3)

 trichothecenes (principally nivalenol, deoxynivalenol, T-2 and

HT-2 toxin)

 zearalenone

 ergot alkaloids, citrinin, sterigmatocystin and alternaria toxins

104
Mycotoxins can cause a variety of adverse health effects in humans
including cancer (some are genotoxic), kidney and liver damage,
gastrointestinal disturbances, reproductive disorders or suppression
of the immune system. Aflatoxins are the most harmful type of
mycotoxin, they can potentially cause cancer or problems with
digestion, reproduction or the immune system.

Mycotoxins are naturally occurring, so their presence in

foods cannot be completely avoided. It is however appropriate to
ensure that controls are in place to ensure that exposure from food
is as low as reasonably achievable. These controls range from
ensuring that good practice is undertaken during growing,
harvesting and storage of foods in addition to establishing
maximum levels where necessary.

LET US SUM UP
The main component of sewage is organic matter (undigested food)
but there are other substances such as oil, heavy metals, nitrogen and
phosphorous compounds (from artificial fertilisers and detergents) which also
have to be removed. Here you will consider the important role of microbes in
the sewage treatment process.

CHECK YOUR PROGRESS

1. Microbve used in cesspool treatment is ----------------

2. The trickling filter treatment is ------------------- method.

GLOSSARY

1. Cesspool – It is a underground tank connected with tube

SUGGESTED READINGS

1. Text Book of Microbiology by Jayaraman Panicker and R.

Ananthanarayan

105
ANSWERS TO CHECK YOUR PROGRESS

1. Bacillus subtilis
2. Aerobic

MODEL QUESTIONS
1. Write detailed notes on largescale waste water treatment.

106
UNIT - 9
INDUSTRIAL APPLICATION OF
MICROBES
STRUCTURE

Overview

Learning Objectives
9.1 Lactic acid production
9.2 Vinegar Production
9.3 Amino acid Production
9.4 Enzyme Production
9.5 Microbes in Energy Generation
Let us Sum up
Check your Progress
Glossary
Suggested Readings
Answers to check your progress
Model questions

OVERVIEW
Microbes, or microscopic organisms, are widely used in large-scale
industrial processes. They are crucial for the production of a variety of
metabolites, such as ethanol, butanol, lactic acid and riboflavin, as well as
the transformation of chemicals that help to reduce environmental pollution.
For instance, microbes can be used to create biofertilizers or to reduce
metal pollutants. Microbes can also be used to produce certain non-
microbial products, such as the diabetes medication insulin. This units
deals with the above said subject.

107
LEARNING OBJECTIVES
To know the industrial usage of microbes in many industrial products.

9.1. LACTIC ACID PRODUCTION

Carbohydrates such as corn and potato starch, molasses and

whey can be used for the production of lactic acid. Let us understand
lactic acid production from whey. Large quantities of whey are
produced during the manufacture of dairy products such as cheese.
Whey contains lactose, nitrogenous substances including vitamins
and salts. Lactobacillus bulgaricus is a bacteria that can grow rapidly
and is capable of converting lactose to lactic acid.

Several important compounds are obtained from

microorganisms such as yeasts and moulds.

These have been summarised in Tables

Table. 2.3 Fungi and its products

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9.2. VINEGAR PRODUCTION

The term vinegar is derived from the French term ‘vinaigre’

which means ‘sour wine’. Vinegar is prepared by allowing wine to get
sour under controlled conditions.

The production of wine involves two types of biochemical

changes:

a. Alcoholic fermentation of a carbohydrate by yeast

Fig. 9.1 Frings method – Vinegar production

b. Oxidation of the alcohol to acetic acid by Acetobacter (i.e. acetic

acid bacteria).

Depending on the carbohydrate used for fermentation, different types

of vinegar are produced. The industrial process of vinegar production
is known as Frings method.

9.3. Amino Acid Production

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 Microorganisms can synthesise amino acids from inorganic
nitrogenous compounds. When the rate of synthesis exceeds the rate
of need of the cell, the excess is secreted into the medium, which can
then be extracted. Lysine is produced using two species of bacteria
namely [Link] and Enterobacter aerogenes in two steps. The first step
involves the formation of DAP or Diaminopimelic acid in the presence
of [Link] under controlled conditions of temperature, aeration and pH.
During the second step, DAP carboxylase obtained from Enterobacter
is added to the culture to convert DAP to lysine.

Lysine is an essential amino acid for humans and since cereal

proteins are deficient in this amino acid, it is used as a supplement for
bread and other food items, (DAP) Glutamic acid, is another amino
acid produced in large amounts by Micrococcus, Arthrobacter sp.
Glutamic acid is a condiment and a flavour enhancing agent in the
form of monosodium glutamate.

9.4 Microbe in Enzymes

Enzymes are biological catalysts necessary to speed chemical

reactions in a cell. This property of enzymes has significance, since it
can be used to speed up industrially important reactions. Also
enzymes are much more efficient than inorganic catalysts since they
can function at room temperature. One more advantage of using
enzymes is that they are substrate specific and produce pure products
which are of significance in pharmaceutical, food and agricultural
industries.

110
 There are however certain disadvantages. Enzymes can be
easily denatured by temperature and pH change and by organic
solvents. They may also be inhibited by products of the reaction. They
are expensive to produce and the choice of the organism is important
since only non-pathogenic organisms should be used. About 200
enzymes are used and most of them are obtained from 11 species of
fungi, 4 species of yeasts and 8 species of bacteria.

Microorganisms are better source of enzymes than plants and

animals because of the following reasons

a. They can be grown in bulk in fermenters under controlled

conditions.

b. They have high growth rates.

c. They can be genetically engineered easily.

d. Mutants can be produced easily.

e. They are easier to maintain as the nutritional requirements are

simpler. They can also be grown on cheap, waste substrates.

f. The quantity of enzymes produced is enormous and they are easier

to recover and purify. The different enzymes and its uses in different
types of industry are summarised in Table. The enzymes obtained are
used in pharmaceutical industry, cheese making, wine making, fruit
juice and wine producers, detergent manufacturers, textile
manufacturers, etc.

Table. 9.1 Enzymes used in Industry

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Immobilised Enzyme Technology

In this technology, the enzyme is bound or immobilised on a

material through which the substance to be changed by the enzyme is
passed. A variety of substances including paper, wood chips, glass
beads and ceramic have been used to immobilise the enzymes.

The main advantages of this technique are as follows

a. The enzymes can be used again and again.

b. The end products can be recovered in pure form.

Microbes and the Pharmaceutical Industry

Compounds of pharmaceutical importance are synthesised by

microbes.

Some important examples are briefly described below

1. Insulin Production

Insulin is a pharmaceutical important compound produced

commercially by [Link] which is genetically engineered. In earlier
times, insulin was isolated from the pancreatic tissue of animals.

112
Human insulin gene is introduced into [Link] by recombinant DNA
technology. The bacterium is grown in large quantities in cultures.

The insulin can be extracted, purified and is ready to use. The

two main advantages of the insulin produced by the recombinant DNA
technology are as follows – (1) it is chemically identical to human
insulin, (2) it can be made available in unlimited quantities.

2. Penicillin Production

The discovery of antibiotics and its commercial production

represent one of the most dramatic case histories in the development
of industrial microbiology. Penicillin was the first antibiotic to be
produced by Alexander Fleming from Penicillium notatum. The
potential of penicillin as an antibiotic was established by Ernest Chain
and Howard Florey. Penicillin was used extensively to treat American
soldiers who were wounded in World War II. Fleming, Florey and
Chain were awarded the Nobel Prize for this discovery.

The major steps in the commercial production of penicillin

a. Preparation of inoculum — A medium of corn liquor, lactose, salts

and other ingredients are mixed, sterilised, cooled and pumped into a
large container known as the fermenter.

b. The mould Penicillium chrysogenum is inoculated into the

fermenter. Sterile air is forced through the fermenter during incubation.

c. After fermentation, the mould mycelium is removed by filtration and

the penicillin produced is extracted in pure form after a series of
processes which includes precipitation, re-dissolving and nitration.

Many antibiotics are produced in a similar manner. The major

differences relate to the organism, composition of the medium and the
method of extraction.

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 Fig. 9.2 Penicillin production

3. Citric Acid Production

Citric acid is an important chemical used in medicines, food and

candies, manufacture of ink, dyeing, etc. Aspergillus niger is the most
commonly used mould for commercial production of citric acid. This
mould is capable of converting sugar into citric acid. Many sugars may
serve as the substrate for the production of citric acid. However,
molasses is generally used.

4. Monoclonal Antibody and Other Bioactive Molecules

Monoclonal antibodies are produced on a commercial scale.

They are particularly significant in treating malignant cells, in immuno-

114
suppression during organ transplantation and for passive
immunisation of specific infectious diseases. They also serve as
analytical reagents for diagnosing cancer and infectious diseases and
hormone assays.

Cyclosporin A is an immunosuppressive agent used in organ

transplant patients. It is produced by a fungus, Trichoderma
polysporum. The statins produced by the yeast Monascus purpureus
is a blood cholesterol lowering agent. It acts as a competitive inhibitor
for the enzyme which is responsible for the synthesis of cholesterol,
thereby stopping its synthesis.

9.5 MICROBES AND ENERGY GENERATION

Microbes have the potential of contributing to energy and fuel

production in several ways. They serve as alternative energy source.

The microorganisms may serve as fuels in one of the following

ways

a. Waste products or plant or microbial biomass may be converted

into liquid or gaseous fuels.

b. Microorganisms may be used to convert solar energy into biomass

and in turn this biomass many be fermented to yield fuels.

c. It may also be possible to use the photo- synthetic membrane

system of the microorganisms to convert solar energy into usable
fuels.

Let us understand this application of microbes depending on the types

of fuel produced.

1. Biogas Production

Biogas is 50-75% methane and the remainder is carbon dioxide with

traces of nitrogen and other gases. Different groups of micro-

115
organisms are used in the process of fermentation. Methanogens are
bacteria used for the production of methane from carbon dioxide and
hydrogen. Methano bacterium is an example of a methanogen.

Methanogens are found in anaerobic sludge produced during

sewage treatment. They are also present in the rumen of cattle. These
bacteria help in the breakdown of cellulose found in the plant food
eaten by the cattle. Animal dung known as ‘gobar’ is rich in these
bacteria. Therefore dung is used for the generation of biogas. This gas
is also known as gobar gas.

Other than animal manures and sewage sludge, food and

domestic wastes, crop remains, paper wastes are also used as
substrates for fermentation. Crops like maize, sugarcane, sugar-beet,
water plants like water hyacinth may also be used.

The overall equation can be represented as follows:

Fig. 9.3 Typical biogas pant

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 The biogas plant in India was built by the Indian Agriculture
Research Institute (IARI) and the Khadi and Village Industries
Commission (KVIC). The biogas plant is a tank which is 10-15 ft deep.
The bio wastes and dung are added into the tank. The gas produced
is collected and sent out through an outlet. The slurry left in the tank
can be used as fertiliser. The biogas generated is used in cooking,
lighting and fuel for car and tractor.

2. Ethanol

Ethanol or ethyl alcohol can be produced by the fermentation of

carbohydrate containing material by microorganisms, especially
yeasts. Ethanol is a valuable alternative fuel resource. The mixture of
gasoline and ethanol is called gasohol and can be used directly in
internal combustion automobile engines. Ethanol and gasoline is
mixed in specific proportions. E10 is the mixture of 10% ethanol and
90% gasoline, while E85 is 85% ethanol and 15% gasoline.

LET US SUM UP
Industrial microbiology includes the use of microorganisms to
manufacture food or industrial products in large quantities. Numerous
microorganisms are used within industrial microbiology; these include
naturally occurring organisms, laboratory selected mutants, or even
genetically modified organisms

CHECK YOUR PROGRESS

1. Lactic acid produced by -------------------

2. ------------------ is the organism used for Citric acid production.

GLOSSARY

1. Insulin – It is an enzyme produced in pancreatic tissue of

animal.

SUGGESTED READINGS

117
 1. Industrial Microbiology by L. E. Casida

ANSWERS TO CHECK YOUR PROGRESS

1. Rhizopus oryzae
2. Aspergillus niger

MODEL QUESTIONS
1. Write about industrial production of enzymes and acids.

118
 Block III

GENERAL VIROLOGY

Unit 10: History of Viruses

Unit 11: Classification and Structure of Virus

Unit 12: Transmission and Isolation of Virus

Unit 13: Life cycle of Virus

Unit 14: Importance of Virus and Special Study

119
UNIT 10

HISTORY OF VIRUS
STRUCTURE

Overview

Learning Objectives
10.1 Origin of Viruses

10.2 Distinguishing Features of Virus

10.3 Early Development of Virology

10.4 Discovery of Virues
Let us Sum up
Check your Progress
Glossary
Suggested Readings
Answers to check your progress
Model questions

OVERVIEW
This unit describes the history of virus and the distinguishing features
of viruses. Viruses were first described as “filterable agents” because their
small size allowed them to pass through filters designed to retain bacteria.
Unlike most bacteria, fungi, and parasites, viruses are obligate intracellular
parasites that depend on the biochemical machinery of the host cell for
replication. In addition, reproduction of viruses occurs by assembly of
individual components rather than by binary fission.

LEARNING OBJECTIVES
To know the origin of visrus and to understand the distingush
features of virus.

120
10.1 ORIGIN OF VIRUSES

Three theories have been put forward to explain the origin of viruses.

These theories highly speculative and are as follows:

(i) Survivors of Pre-Cellular First Living Inhabitants of the Earth:

This theory intimately rests on the theory of origin of life on Earth. Life,
according to this theory, originated from simple inorganic compounds by a
slow biochemical evolution of “ordinary” chemical reactions spread over
millions and millions of years.

It is speculated that during the course of origin of life on Earth

somewhere at the stage when complex chemical molecules united to form
still more complex molecules which could mate with still other metastable
molecules till a relatively large molecule (like nucleoproteins) capable of
growth and division, a simple virus or a protovirus may have originated. This
theory, however, enjoys some insurmountable objections. Present day
viruses are all obligate parasites and it is difficult to conceive of their origin
before the origin of their hosts (cells) which are at a far higher scale of
evolution.

Viruses use the same genetic code as cellular organisms and

depend solely and entirely on ribosomes, transfer RNAs and enzymes of the
host cell for protein biosynthesis. Moreover, viral nucleic acid has the same
properties and the same mode of replication as the nucleic acid of cellular
organisms.

Viruses are considered to have originated by retrogressive evolution

from free-living cells, according to this theory. A parasite evolves
retrogressively as it takes the readymade metabolites from its host instead of
synthesizing them himself.

If the parasite continues to evolve retrogressively then, to save labour

and energy it would slowly loss some of its physiological, morphological and
even genetical functions that become super numerary in its new ecological

121
niche and new mode of biological existence. A parasite would, therefore get
regressed to a much simpler organism.

Obligate intracellular parasitism is the most specialized type of

parasitism and such a parasite would ultimately possess only the bare
minimum and this minimum is the possession of a nucleic acid (to ensure
genetic continuity) enclosed within a protein shell (to ensure safety of the
nucleic acid).

Shedding of all unnecessary morphological, physiological and

genetical materials would necessarily reduce the size. A formerly free-living
organism would thus be transformed into a virus. Many virologists support
the theory of retrogressive origin of viruses but the same is opposed also by
others.

Derived from Normal Constituents of the Cell

An eukaryotic cell possesses organelles like chloroplasts and

mitochondria which are self-replicatine semi- autonomous structures and
reproduce their like. Chloroplasts and mitochondria increase in size and then
divide while kinetosome is synthesized near a pre-existing one by assembly
from the tubular materials.

Besides they possess DNA which is functional and possesses its

own mutational history, codes for the synthesis of mRNA and also
presumably for protein. Mitochondria mutate to non-functional forms in
Neurospora and yeasts, while chloroplasts mutate to undeveloped,
colourless proplastids in algae and higher plants. These mutations are based
on genetic changes. Cells also contain certain organelles that exceptionally
undergo autonomous unrestricted replications; examples are the centrioles
in Marsilea and sperms and nuclear genes in the amphibian oocytes.

Viruses resemble above mentioned cellular organelles in chemical

composition, possession of nucleic acid, genetic continuity of genome,
independent mutational history, capacity to replicate independently under
their own genetic control and also under the overall regulatory control of and
within the premises of the host cell.

122
 Many of the cellular organelles or factors possess some of the
distinctive characters of viruses, or more specifically, of viral genetic
determinant (reproductive independence, evolutionary independence,
independent cell to cell transfer and infectivity and pathogenicity) while
others could be conferred on them by a specific arrangement of nucleotides.
Viruses could, therefore, be derived from any or several of these cellular
components and it is possible that different viruses have originated
differently.

Some of the possibilities are given below

1. Primordial self-replicating molecules may have mutated to regain the

capacity to enter (‘infect’) the cell and then integrate with it. Such a molecule
would be a ‘virus’.

2. Some plasmids or even chromosomal segments may have evolved by

merging of some primitive self- replicating molecules with the cellular
genome. If this is true then it is conceivable that some of the genes or
groups of genes may revert to their ancestral habit and may have
regained/evolved the genetic independence and independent transfer of this
genetic material. This would result in the ‘virus’.

3. Some genes of the cell could have escaped of the control mechanisms of
the cell and may have acquired the capacity of autonomous replication
independent of the division of the cell and capacity of independent transfer.
Integration of these genes with the host genome would give us a prophage.
Origin of bacteriophage from such prophage DNA has been outlined by
Lindegren in 1962. It is suggested by some that bacteriophages containing
DNA may have evolved from a number of genetic transfer elements (like F
factor, bacteriocinogenic factor, etc.) occurring in the prokaryotic cells.

4. DNA viruses of eukaryotic cells may have originated from the functional
DNA of cellular organelles (e.g., mitochondria and chloroplast) rather than
for nuclear DNA.

5. Origin of DNA plant viruses is somewhat more difficult to understand since

there is no definite information with respect to the integration of plant viral

123
nucleic acid into the genome of the plant cell. There is, however, some
suggestive evidence in this connection. Some experimental evidence
suggests that cut surfaces of barley seeds take up the DNA of the bacterium
Micrococcus lysodeikticus and that it integrates into nuclear DNA of barley
and replicates.

In short, therefore, viruses may have originated from cell constituents

which escape the control mechanisms of the cell, regained/developed the
capacity of autonomous self-replication and ability to mediate their own
independent cell to cell transfer and could enter or infect cells to which they
did not belong.

10.2. DISTINGUISHING FEATURES OF VIRUS

(i) All viruses are obligate parasites,

(ii) They multiply only within their living hosts cells and remain metabolically
inert outside the host cell,

(iii) They are ultramicroscopic and can only be viewed with electron
microscope (the smallest known virus is merely 0.002 µm in diameter, while
the largest ones are typically about 0.8 µm in diameter),

(iv) Viruses are, actually, nucleoproteins. The proteinaceous coat (capsid)

surrounds the nucleic acid, which forms the central core of the virus
particles,

(v) The viral genetic material, the nucleic acid, may be either DNA or RNA.
The two nucleic acids are never present in a given virus,

(vi) These are the nucleic acids of the viruses which are infectious, and not
the protein coat,

(vii) Viruses are usually so minute that they can easily pass through a filter,
which can hold back even the smallest bacteria,

(viii) Viruses are easily transmitted from infected host to the healthy ones
through various agencies,

124
(ix) Viruses are so effective that even their smallest amount can cause
infection on the host successfully,

(x) Viruses can easily be crystallized,

(xi) Viruses are considered to be host-specific and represent obligate

parasitism even at genetic level, and

(xii) Since the viruses have no metabolic activities of their own and utilize the
metabolism of host cells, antibiotics have no effect on them.

10.3 EARLY DEVELOPMENT OF VIROLOGY

Literature reveals that ancient people were well acquainted with

today’s viral diseases such as small pox, cowpox, rabies, measles, etc.
without any knowledge of the nature of the diseases. There is some
evidence that the Roman Empire was severely weakened and declined due
the great epidemics of measles and smallpox diseases that appeared during
A.D. 150 to 180 and A.D. 251 to 266. Aztec Empire in Mexico was captured
by Hernan Cortes, a Spanish, in the 1520s. It is considered that this
conquest was made possible by smallpox epidemic that ravaged Mexico city.

Early Prevention of Viral Diseases

Prevention of viral diseases by immunization progressed years

before the discovery of viruses. This practice was used in Asia for centuries
to produce immunity to smallpox before it was introduced in England .She
observed that Turkish women inoculated their children against smallpox by
material taken from a pustule of an infected person. The material was
scratched into the skin of the children to be immunized. In most cases this
resulted in a mild case of smallpox without he scarring that was common in
naturally acquired cases.

The immunization against smallpox was practiced without any

explanation of how or why it worked. Later during the end of the century an
English country doctor Edward Jenner, stimulated by a girl’s claim that she
could not catch smallpox because she had had cowpox, reported to Royal

125
Society in London the value of immunization with cowpox as a means of
protecting against smallpox; a clear case of vaccination.

This he did on the basis of the fact that when he inoculated a 8-year
old boy, James Phipps, with cowpox content, the boy escaped from smallpox
infection. Jenner then began inoculating humans with material from cowpox
lesions, and published the results of 23 successful vaccinations in 1798.
Jenner’s explanation regarding cowpox vaccination against smallpox
established the scientific credibility of vaccination to prevent disease and
was accepted by the scientists and physicians of the time.

Jenner, therefore, is credited to have developed first vaccine from

cowpox (Latin name Vaccinia). Later on, Louis Pasteur developed vaccines
(attenuated microorganisms) against chicken cholera and anthrax in 1880,
and against rabies in 1885.

10.4 DISCOVERY OF VIRUSES

The tobacco crop in Holland was struck by a severe disease around

1870. Adolf Mayer, Director of Agricultural Experimental Station,
Wageningen, began his studies on this disease about 1880 and published
his results in 1886.

Mayer christened the disease as “MOSAIKKRANKHEIT” (mosaic-

like), from the mosaiclike pattern on leaves of diseased plants and
succeeded in reproducing the disease by infecting juice extracted from
infected tobacco leaves onto healthy ones; he could not succeed in
identifying the real agent that caused the disease.

However, Mayer’s contribution will always be remembered as he was

the first person who put first step forward in the development of a new
discipline later recognised as ‘Virology’. The development of the porcelain
bacterial filter in 1884 by Charles Chamberland, one of the Pasteur’s
collaborators and the discoverer of autoclave, made possible the discovery
of what are now called viruses.

126
 Chamberland’s filter was first used in the study of tobacco mosaic
disease in the year 1892 when D. Ivanowski first successfully experimentally
demonstrated that the tobacco mosaic disease has been caused by agents
which successfully passed the Chamberland-filter that retains even the
smallest bacteria.

It was an important clue, but contrary to his experimental result and

despite his inability to isolate any bacterium, Ivanowski still maintained that
either the ‘pathogenic bacterium’ somehow passed through the filter or a
‘toxin’ secreted by them passed through the filter and made the filtrate
infectious. Within six years after the experiments of Ivanowski, M. Beijerinck
(1898) confirmed by repeating the same experiments and found that the
tobacco mosaic disease was caused not by any pathogenic bacteria or toxin
but rather by some new type of pathogenic agents which he called
“contagium vivum fluidum” (infectious living fluid) and referred subsequently
to it as a “virus” (poison).

He observed that the viruses multiply only inside the living cell, but
can survive for long periods in a dried state. At the same time F. Loeffler and
Paul Frosch in Germany demonstrated that the hoof and mouth disease of
cattle was also caused by a filterable virus rather than by a toxin. W. Reed
(1900) studied the yellow fever disease spreading in Cuba and
demonstrated that this human disease was due to a filterable virus that was
transmitted by mosquitoes.

Mosquito control shortly reduced the severity of the yellow fever

disease. Thus by the beginning of 20th century, it has been established with
certainty that filterable viruses were independent intities different from
bacteria and were able to cause diseases in plants, animals, and humans.

Post-Discovery Contributions

After the discovery of viruses as independent intities existing in the

biological world, certain important developments were made in the field of
virology. In 1908, V. Ellerman and O. Bang reported lukemia as a viral
disease. Peyton Rous reported in 1911 that a virus was responsible for a

127
malignant muscle tumour in chickens. Discovery of bacteriophages, the
viruses that attacked bacteria, was another milestone in the development of
virology.

The first published observation suggesting that the bacteria

themselves also could be attacked by viruses was made in 1915 by F.W.
Twort. Twort observed that the colonies of micrococci and intestinal bacilli
bacteria were lysed and the lytic effect spread from one colony to the other.
He speculated that the lysis of bacterial colonies would have been due to a
virus.

Twort did not follow up these observations and it remained for Felix
d’Herelle to establish decisively the existence of bacterial viruses. D’Herelle
isolated bacterial viruses from patients with dysentery, probably caused by
Spigella dysenteriae.

He observed that when a virus suspension was spread on a layer of

bacteria growing on agar, clear circular areas containing viruses and lysed
bacterial cells appeared. D’Herelle demonstrated that bacterial viruses could
reproduce only in live bacterial cells, and named these viruses
‘bacteriophages’ because could eat bacterial cells creating clear areas (the
plaques) in bacterial “lawns”.

Establishment of chemical nature of viruses was a very significant

contribution in the field of growing virology. W.M. Stanley (1935) first
crystallized the tobacco mosaic virus (TMV) and found it to be largely or
completely protein.

Shortly later, F.C. Bawden and N.W. Pirie separated tobacco mosaic
virus particle into protein and nucleic acid. Thus by the late 1930s it was
established with certainty that the viruses were made up of nucleic acids and
proteins, and could reproduce only in living cells.

LET US SUM UP
Viruses are noncellular parasitic entities that cannot be classified
within any kingdom. They can infect organisms as diverse as bacteria,

128
plants, and animals. In fact, viruses exist in a sort of netherworld between a
living organism and a nonliving entity. Living things grow, metabolize, and
reproduce. In contrast, viruses are not cellular, do not have a metabolism or
grow, and cannot divide by cell division. This unit deals the history of virus.

CHECK YOUR PROGRESS

1. Virus are --------------

2. ----------------------- named tobacco disease as

MOSAIKKRANKHEIT

GLOSSARY

1. Capsid – Protenecious coad sarrounds viral nucleic acid.

SUGGESTED READINGS

1. Basic Virology by Edward K. Wagner

ANSWERS TO CHECK YOUR PROGRESS

1. obligate parasites
2. Adolf Mayer

MODEL QUESTIONS
1. Write abou the origin of virus.

129
UNIT 11

CLASSIFICATIONS AND STRUCTURE

OF VIRUS

STRUCTURE

Overview

Learning Objectives
11.1 On the basis of type of host

11.2 On the basis of Nucleic acid

11.3 Harrison Classification

11.4 The Baltimore Classification

11.5 Structure of Viruses

Let us Sum up
Check your Progress
Glossary
Suggested Readings
Answers to check your progress
Model questions
OVERVIEW
A virus classification based on nucleotide sequences is actually a
phenotypic classification with the discriminating characters being molecular
rather than morphological, physiological, or relational

LEARNING OBJECTIVES
To understand the structure and identify the virus by classification.

130
Classification of Viruses

Viruses may be classified according to the type of the host, genetic

material and number of strands.

11.1 ON THE BASIS OF TYPE OF HOST

1. Animal Viruses

They live inside animal cells including man. On entering the cell,
these disturb the metabolism of the host cell and cause various diseases.
The common animal viruses are small pox virus, influenza virus, mumps
virus, polio virus and herpes virus. In many animal viruses an extra envelope
surrounds their protein coat. The membrane consists of proteins, lipids and
carbohydrates and is derived from the host plasma membrane.

Animal viruses may enter cells by attaching to the surface. Some are
then engulfed by the cell through pinocytosis or phagocytosis. In such cases,
uncoating of the viral nucleic acid might occur within the cell. Inside the host
cell they may multiply and form numerous new viral particles. Usually, animal
viruses release from the host cells by the rapturing and subsequent death of
the host cells.

2. Plant Viruses

They are parasites of plant cells. Their genetic material is RNA which
remains enclosed in the protein coat. The most important plant viruses are
tobacco mosaic virus (TMV), tobacco rattle virus (TRV), potato virus (PV),
southern bean mosaic virus (SBMV), beet yellow virus (BYV) and turnip
yellow virus (TYV).

3. Bacterial virus:

They are parasitic on bacteria and so also called bacteriophages.

There are many varieties of bacteriophages. Their size and shape varies
from species to species. Some phages are spherical, some comma-shaped
whereas majority of them have tadpole-like appearance.

11.2 ON THE BASIS OF NUCLEIC ACIDS

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1. DNA viruses:

These viruses possess DNA as the genetic material. On replication this DNA
produces new DNA. DNA transmits information for protein synthesis through
RNA. (DNA → RNA → PROTEIN).

2. RNA viruses

These viruses possess RNA as the genetic material. The RNA

replicates directly to produce new RNA. Information for protein synthesis
passes from RNA to protein without involment of DNA. (RNA → RNA →
PROTEIN).

3. DNA – RNA viruses

In a group of RNA tumour viruses called leukoviruses or rousviruses

the genetic material is alternately DNA and RNA. In addition to the normal
mode of transfer found in DNA viruses (DNA → RNA → PROTEIN) the
rousviruses also transfer information from RNA to DNA (RNA-DNA-RNA -
PROTEIN).

With respect to number of strands, four types of nucleic acids have

been found in viruses

1. Double stranded DNA

Double stranded DNA has been reported in pox viruses, the

bacteriophages T 2, T 4, T 6, T 3, T 7 and lamda, herpes viruses, adeno
viruses, polyoma virus SV-40 and papilloma viruses.

2. Single stranded DNA

Single stranded DNA is found in the bacteriophages ph i X 174 and

M-13 and is cyclic.

3. Double stranded RNA

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 Double stranded RNA has been found within viral capsid in the
reoviruses of animals and in the wound tumour virus and rice dwarf viruses
of plants.

4. Single stranded RNA

Single stranded RNA is found in most of RNA viruses e.g. Tobacco

mosaic virus, influenza virus, poliomylitis bacteriophage MS – 2, F – 2,
Coliophage R 17 and the avian leukemia virus.

11.3 HARRISON CLASSIFICATION

On the basis of the behaviour of virus in the hosts, vector relations,

particle properties and particle composition, Harrison et al. (1971) divided
plant Viruses into 16 groups, as detailed below:

1. Tobravirus group. Type member

133
Tobacco rattle virus.

2. Tobamovirus group. Type member

Tobacco mosaic virus

3. Potexvirus group. Type member

Potato virus X

4. Caria virus group. Type member

Carnation latent virus

5. Poty virus group. Type member

Potato virus Y

6. Cucumovirus group. Type member

Cucumber mosaic virus.

7. Tymovirus group. Type member:

Turnip yellow mosaic virus.

8. Comovirus group. Type member

Cowpea mosaic virus

9. Nepovirus group. Type member

Tobacco ringspot virus.

10. Bromovirus group. Type member:

Brome mosaic virus.

11. Tombus virus group. Type member

Tomato bushy stunt virus.

12. Caulimovirus group. Type member

Cauliflower mosaic virus.

13. Alfalfa mosaic virus. Type member

Alfalfa mosaic virus.

134
14. Pea Enation Mosaic virus. Type member

Pea enation mosaic virus.

15. Tobacco necrosis virus. Type member

Tobacco necrosis virus A strain.

16. Tomato spotted wilt virus. Type member

Tomato spotted wilt virus.

The greatest drawback of this classification is that only the well-

studied viruses have been placed in the above mentioned 16 groups and
many little known viruses have been left. In addition, viruses resembling
wound tumour and lettuce necrotic yellows have also not been placed in
these groups.

11.4 THE BALTIMORE CLASSIFICATION

The Baltimore system of virus classification provides a useful guide

with regard to the various mechanisms of viral genome replication. The
central theme here is that all viruses must generate positive strand mRNAs
from their genomes, in order to produce proteins and replicate themselves.
The precise mechanisms whereby this is achieved differ for each virus
family. These various types of virus genomes can be broken down into
seven fundamentally different groups, which obviously require different basic
strategies for their replication. David Baltimore, who originated the scheme,
has given his name to the so-called "Baltimore Classification" of virus
genomes. By convention the top strand of coding DNA written in the 5' - 3'
direction is + sense. mRNA sequence is also + [Link] replication
strategy of the virus depends on the nature of its genome. Viruses can be
classified into seven (arbitrary) groups:

I: Double-stranded DNA

[Link]

[Link]

135
 [Link]
II: Single-stranded (+)sense DNA

[Link]
III: Double-stranded RNA

[Link];

[Link])
IV: Single-stranded (+)sense RNA

[Link];

[Link],

V: Single-stranded (-)sense RNA

[Link],

[Link],

VI: Single-stranded (+)sense RNA with DNA intermediate in life-cycle

[Link]
VII: Double-stranded DNA with RNA intermediate

[Link]

11.5 STRUCTURE OF VIRUSES

Viruses were first described as “filterable agents” because their small

size allowed them to pass through filters designed to retain bacteria. Unlike
most bacteria, fungi, and parasites, viruses are obligate intracellular
parasites that depend on the biochemical machinery of the host cell for
replication. In addition, reproduction of viruses occurs by assembly of
individual components rather than by binary fission.

Virus is defined as “Viruses are acellular organisms whose genomes

consist of nucleic acid, and which obligately replicate inside host cells using
host metabolic machinery and ribosomes to form a pool of components
which assemble into particles called VIRIONS, which serve to protect the
genome and to transfer it to other cells.”

136
 Viruses are infectious agents with both living and nonliving
characteristics. They can infect animals, plants, and even other
microorganisms. Viruses that infect only bacteria are called bacteriophages
and those that infect only fungi are termed mycophages . There are even
some viruses called virophages that infect other viruses.

Fig.11.1: A Virus

Structure

The basic structure of a virus is made up of a genetic information

molecule and a protein layer that protects that information molecule. The
arrangement of the protein layer and the genetic information comes in a
variety of presentations. The core of the virus is made up of nucleic acids,
which then make up the genetic information in the form of RNA or DNA. The
protein layer that surrounds and protects the nucleic acids is called the
capsid. When a single virus is in its complete form and has reached full
infectivity outside of the cell, it is known as a virion. A virus structure can be
one of the following: icosahedral, enveloped, complex or helical.

All viruses contain the following two components:

1) a nucleic acid genome and

2) a protein capsid that covers the genome. Together this is called

the nucleocapsid. In addition,

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3) lipid envelope. The entire intact virus is called the virion. The structure and
composition of these components can vary widely.

Viral Genomes: While the genomes of all known cells are comprised of
double stranded DNA, the genomes of viruses can be comprised of single or
double stranded DNA or RNA. They can vary greatly in size, from
approximately 5-10 kb (Papovaviridae, Parvoviridae, etc.) to greater than
100-200 kb (Herpesviridae, Poxviridae). The known structures of viral
genomes are summarized below.

DNA: Double Stranded - linear or circular

Single Stranded - linear or circular

Other Structures - gapped circles

RNA: Double Stranded - linear

Single Stranded - linear : These single stranded genomes can be

either + sense, - sense, or ambisense The sense strand is the one that
can serve directly as mRNA and code for protein, so for these viruses, the
viral RNA is infectious. The viral mRNA from - strand viruses is not
infectious, since it needs to be copied into the + strand before it can be
translated. In an ambisense virus, part of the genome is the sense strand,
and part is the antisense.

The genome of some RNA viruses is segmented, meaning that a virus

particle contains several different molecules of RNA, like different
chromosomes.

Icosahedral Symmetry
A body with cubic symmetry possesses a number of axes about
which it maybe rotated to give a number of identical appearances. The
occurrence of icosahedral features in quite unrelated viruses is not a matter
of chance, but a preference. An ICOSAHEDRON is composed of 20 facets,
each an equilateral triangle, and 12 vertices, and because of the axes of
rotational symmetry is said to have [Link] symmetry. There are, in fact, six 5-
fold axes of symmetry passing through the vertices, ten 3-fold axes

138
extending through each face and fifteen 2-fold axes passing icosahedral
capsids with [Link] symmetry are built up by using 3 identical subunits to form
each triangular face, thereby requiring 60 identical subunits to form a capsid.
A few virus particles are constructed in this way, e.g. bacteriophage ØX174.
Each unit would be related identically (equivalent) and asymmetrically with
its neighbours, and none of the units would coincide with an axis of
symmetry.

Helical symmetry

Tobacco mosaic virus (TMV) is representative of one of the two

major structural classes seen in viruses of all types, those with helical
symmetry. The simplest way to arrange multiple, identical protein subunits is
to use rotational symmetry & to arrange the irregularly shaped proteins
around the circumference of a circle to form a [Link] discs can then be
stacked on top of one another to form a cylinder, with the virus genome
coated by the protein shell or contained in the hollow centre of the cylinder.
Closer examination of the TMV particle by X-ray crystallography reveals that
the structure of the capsid actually consists of a helix rather than a pile of
stacked disks.

Bacteriophage

Complex These virus structures have a combination of icosahedral

and helical shape and may have a complex outer wall or head-tail
morphology. The head-tail morphology structure is unique to viruses that
only infect bacteria and are known as bacteriophages. The head of the virus
has an icosahedral shape with a helical shaped tail. The bacteriophage uses
its tail to attach to the bacterium, creates a hole in the cell wall, and then
inserts its DNA into the cell using the tail as a channel. The Poxvirus is one
of the largest viruses in size and has a complex structure with a unique outer
wall and capsid. One of the most famous types of poxviruses is the variola
virus which causes smallpox.

139
 Fig.11.2: Virus morphology

LET US SUM UP
Viruses are small obligate intracellular parasites, which by definition
contain either a RNA or DNA genome surrounded by a protective, virus-
coded protein coat. Viruses may be viewed as mobile genetic elements,
most probably of cellular origin and characterized by a long co-evolution of
virus and host.

CHECK YOUR PROGRESS

1. --------------- are douvle stranded DNA virus
2. TMV is single strandad ---------------------

GLOSSARY
1. Nucleocapsid- a progtein cover of genome

SUGGESTED READINGS
1. Viruses by Susan Payne

140
ANSWERS TO CHECK YOUR PROGRESS
1. Phages
2. RNA virus

MODEL QUESTIONS
1. Wrtie detailed accouon Virus structure

141
UNIT 12

TRANSMISSION OF VIRUSES
STRUCTURE

Overview

Learning Objectives
12.1 Mechanical Transmission
12.2 Vegetative and Graft Transmission
12.3 Pollen Transmission
12.4 Seed Transmission
12.5 Nematode Transmission
12.6 Fungal Transmission
12.7 Insect Vector Transmission
12.8 Dodder Transmission
12.9 Isolation of Viruses
Let us Sum up
Check your Progress
Glossary
Suggested Readings
Answers to check your progress
Model questions

OVERVIEW
The transmission of virus happened in many ways such as
mechanical, vegetative, pollen, seed, nematode, fungal, insect vector and
dodder media. Transmission of virus takes place quick and fast. The
transmission of virus has been studied in this un it.

LEARNING OBJECTIVES
To understand the transmission of virus

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12.1 MECHANICAL TRANSMISSION

In nature plant viruses are mechanically transmitted from diseased to

healthy plants by rubbing leaves together, injecting plant extract, by action of
animals, etc. Viral particles remain adhered to plant surfaces, epidermis or
hairs. During rubbing the cells are broken and viral particles are liberated in
the damaged cells. Transmission through this mechanism occurs in such
plants which are closely planted. Similarly viral particles attached on surface
of animal body are transmitted when they rub their body first on infected
plants and then on healthy plants. Viral particles enter through the injuries
made by animals. Similarly birds also transmit viruses by this method, for
example TMV.

Potex virus survives on human clothing and agricultural tools such as

cutting knives, sickles, etc. These tools are frequently used in agriculture and
horticulture. Hence their use first on infected plants then healthy plants
facilitates viral transmission.

Indicator Plants

The test plants that react promptly and characteristically to particular

viruses are called ‘indicator plants’ or differential hosts’. Only susceptible
plants act as indicator plant but not the virus resistant plants.

Usually the cotyledons of cucumber and primary leaves of cowpea

and beans are inoculated by viruses, because older plants are less
susceptible. Virus resistant plants consist of some special inhibitory
substance that prevent the transmission of viruses. Table shows some of the
indicator plants commonly used for inoculation purpose.

12.2 VEGETATIVE AND GRAFT TRANSMISSION

Generally viral particles are present almost in all parts of

systematically infected plants. Virus will certainly be transmitted to the
progeny, if any part of such mother plants in used for vegetative propagation
through rhizomes, bulbs, corns, tubers, cuttings, etc. Therefore, one must

143
not use the infected vegetative part of vegetatively propagating plants such
as dahlia, chrysanthemums, carnations, potatoes, etc.

Grafting technique (placing the cut end of one plant onto immediate
contact of tissues of other plants to establish a union product in one plant)
has been well practiced in India since time immemorial. There is wide variety
of grafting techniques such as stem grafting or wedge grafting, tuber grafting
(in potatoes), root grafting, etc. Grafting is widely used commercially for
propagation of plants.

For example, more than 4,000 varieties of mango have been possible
due to grafting. Systemic virus can be transferred from infected portion of a
plant to the healthy portion of other one e.g. colour breaking of tulip, apple
mosaic. Nicotiana glutinosa plants die when tomato or tobacco plants
systematically infected with TMV are grafted with it because TMV produces
necrosis of leaves and buds.

12.3 POLLEN TRANSMISSION

When pollens consisting for viruses fall on stigma of female plants,

they germinate and eventually facilitate the virus to infect the ovules of
plants. Such viruses are called pollen-borne viruses. Example of pollen-
transmitted viruses are: barely stripe virus, tobacco ring spot virus, bean
common mosaic virus, fruit ring spot virus. Dhatura mosaic virus is
transmitted through pollen to seeds in 79% offspring.

12.4. SEED TRANSMISSION

Seed transmission of viruses is very rare but many viruses are known
to be seed transmitted. However, a very low level (0.1%) of seed
transmission had epidemiologically been found out.

Some example of seed transmitted viruses are bean mosaic virus,

tomato ring spot virus, tobacco ring spot virus, cowpea mosaic virus,
cucumber mosaic virus, mung and urd bean mosaic viruses, etc. Seed
transmitted viruses are present in embryo, endosperm or seed coat. After
seed germination, virus- infected seedlings are produced.

144
 A substantial yield loss is caused by viruses. For example, soya bean
mosaic virus results in 10-20% decline in seed weight with seed
discolouration. Since seeds are carried for a long distance within a country
or across the country, viruses are transmitted over long distances. Seed-
borne viruses get good chance of survival between two seasons and during
adverse conditions. Besides, nematodes also transmit the seed-borne
viruses.

12 5. NEMATODE TRANSMISSION

There are some plant parasitic nematodes that feed roots of plants.
Such nematodes also act as vector for some viral pathogens. Vectors are
the organism that assist in transmission of viruses. Examples of some plant
parasitic nematodes are: Longidorus, Paratrichodorus, Trichodorus and
Xiphinema. These nematodes transmit viruses for about 10 months but
transmission does not involve viral replication inside the vector.

Fig. 12.1 Virus transforming nematodes

There is virus-vector specificity for transmission by nematodes.

Nepoviruses (polyhedral) are transmitted by Xiphenema and Lingidorus, and
Tobraviruses (tubular) are transmitted by Paratrichodorus and Trichodorus.

In soil two types of virus transmission can be observed

(a) Some of the viruses remain active in debris of roots are leaves. Healthy
plants that come in contact of debris are infected, e.g. TMV,

(b) Virus is transmitted to healthy plants by nematodes that inhabit the soil
e.g. potato rattle virus.

145
12.6. FUNGAL TRANSMISSION

There are many soil-inhabiting fungi which transmit several viruses,

for example Olpidium brassicae, Polymyxa graminis, Synchytrium
endobioticum and Spongospora subterranean. These fungi are obligate
endoparasite of higher plants.

Their zoospores infect the roots of new hosts, introduced viruses and
produce virus specific symptoms. These fungi acquire virus from virus-
infected plants which persists in soil for several months or years. O.
brassicae transmit tobacco necrosis virus, P. graminis transmit wheat
mosaic virus and S. subterranean transmit potato mop top virus.

12.7 INSECT VECTOR TRANSMISSIONS

Vectors are highly mobile and play an important t role in natural

ecology of viruses. In many cases relationship between virus and vector is
an intimate biological association, besides mere mechanical transfer. Most of
the viruses are transmitted by vectors. They have slender, needle-like
mouthparts to which they pierce the cells and suck cell sap of host plants.
Virus is transmitted to healthy plants when the viruliferious insects feed the
plant tissue.

Viruses are divided into three groups, on the basis of length of the
period and relationship with insect

(a) Non-persistent [where acquisition period i.e. feeding period on diseased

plant is for a short period (10-60 second); thereafter rate of transmission
decreases because viruses survives for a short period],

(b) Semi-persistent (acquisition period is of 12-24 hours and transmission

occurs immediately after acquisition for 1-3 hour.

Hence, virus survives for a few hours in the vectors), and (c) persistent
(viruses survives for a week or month in the vectors; hence, transmission
occurs only where the inoculation feeding lasts at least for some hours).

Some of the important insect vectors and viruses transmitted by them

are described in this section

146
(i) Aphids

Aphids are the most notorious and important groups of plant vector.
They are found in large numbers during spring and winter seasons. They
show preference towards feeding of the hosts, for example Aphis craccivora
and A. fabae prefers beans; A gossypii prefers cotton, cucurbits, chilli and
brinjal; Myzus persicae prefers tobacco; Liaphis erysimi infects crucifers
such as mustard.

Examples of viruses transmitted by aphids are: barely yellow dwarf virus,

potato virus S, alfalfa mosaic virus, cucumber mosaic virus, potato leaf roll
virus, lettuce necrotic yellow virus, and bean mosaic virus, barely mosaic
virus, pea mosaic virus, chilli mosaic virus, etc.

(ii) Leafhoppers

All the leafhopper-borne viruses are transmitted in a persistent

manner, except rice tungro virus and maize chlorotic dwarf leaf virus which
are transmitted in semi-persistent manner. Examples of viruses transmitted
by leafhoppers are: maize streak virus by Cicardulina mobila, rice dwarf virus
by Nephotettix nigripictus, beet curly top virus by Circulifer tennelus, etc.

(iii) Grasshoppers

Grasshoppers also transmit many viruses such as TMV, turnip yellow

mosaic virus, turnip crinkle virus, etc.

(iv) Beetles:

More than seventy four species of beetle are known to transmit virus.
Examples of beetle-transmitted viruses are cow pea mosaic virus, turnip
yellow mosaic virus, squash mosaic virus, southern bean mosaic virus.

12 8. DODDER TRANSMISSION

Dodders are the trailer or climber parasitic plants which grow forming
bridge between two plants. Cuscuta reflexa is the most famous dodder plant
that lacks leaves. They belong to the family Convolvulaceae.

147
 Dodders wind around the host and penetrate its haustoria into host
tissue sending up to vascular tissue. Haustoria acquire virus from the
infected plants that are eventually transmit to the new hosts. Dodder
transmitted viruses are sugar beet curly top virus, tomato bushy stunt virus,
tobacco rattle virus, etc.

12.9 ISOLATION OF VIRUSES

Viruses are isolated from infected host cells containing mature

virions. The cells are mechanically disrupted and the cell contents are
released in a suitable buffer solution. The suspension containing the virions
and cell ingredients is then subjected to centrifugation for several times at
different speeds to fractionate the virions from other cell components. Such
procedure is known as differential centrifugation. A more refined technique is
the density gradient centrifugation which is usually applied for getting a more
purified sample of viruses. Before subjecting the sample of virus to density
gradient centrifugation, it is initially purified by differential centrifugation. A
density gradient is prepared in a centrifuge tube. For example, a sucrose
gradient contains a linearly increasing concentration of sucrose from top to
bottom of the centrifuge tube. The partially purified virus sample is poured on
the top and the tube is subjected to high-speed centrifugation for several
hours in an ultracentrifuge. The centrifugal force drives the viral particles
towards the bottom until they settle at a density gradient of sucrose which
equals that of the virions, forming a concentrated zone or band. The
suspension of virions can then be removed from this band with the help of a
pipette. The bacteriophages causing lytic infections can be isolated by a
more or less similar method by differential centrifugation of the lysate to
eliminate cell debris and the non-lysed intact cells of the host bacteria.

Assay of Viruses

Viral assay means determination of number of viral particles per unit

volume of a sample. Several methods are available for this purpose. The
total number of viral particles in a sample including both viable and non-
viable virions can be counted directly by means of electron microscopy.

148
 For this purpose, a known volume of a purified sample of the virus is
mixed with a known volume of a suspension of minute polystyrene latex
beads of known concentration. The mixture is sprayed in droplets on a
supporting membrane, dried and shadowed.

From the ratio of the number of beads and that of viral particles the
number of virions per unit volume can be calculated, when the preparation is
examined under an electron microscope. For example, if in a preparation, a
droplet reveals presence of 200 viral particles and 20 latex beads, the
concentration of virions / ml would be 200/20 multiplied by the number of
beads per ml in the suspension.

If the bead concentration in the original suspension is 5 x 1010 per ml.

The viral count would be 200/20 x 5 x 1010/ml = 5 x 1011/ml. The
concentration of viruses in a sample is also known as its titre. Among the
other methods for assay of viral titres in a sample, the plaque method is a
very important one.

It makes use of the infectivity of the viruses and their capacity to

destroy the infected cells. The plaque method was first developed for
detection and counting of bacteriophages and later it was extended to the
study of animal viruses. The plaque method is widely used for its simplicity,
accuracy and reproducibility.

For assay of bacteriophages, a sample is diluted many-fold (~10-20 to

10-25) and mixed with a drop of a dense liquid culture of the host bacterium
and a few milliliters of molten soft agar medium at 44°C. The mixture is
poured on the surface of an already solidified hard nutrient agar in a plate
and uniformly spread to allow the phages and bacteria to be evenly
distributed.

Overnight incubation of the plates reveals the presence of a number

of clear areas, known as plaques, on a lawn of continuous growth of the host
bacterium. Plaques are formed due to infection and destruction of the host
cells producing the clear areas. The number of plaques is proportional to the
concentration of the virus.

149
 Each plaque is produced by a plaque-forming unit (PFU). Thus, if an
aliquot of 0.1 ml of a 10-20 dilution of the phage sample is plated and produce
an average of 40 plaques per plate, the titre is 40/0.1 x 1020PFU/ml. It should
be noted that the number of PFUs cannot be taken as equal to the number
of phages, but the two are proportional to each other.

The plaque method with necessary modification has also been used
for assay of animal viruses. In place of bacteria, a suspension of cultured
animal cells is used as host. The bacteriological nutrient medium is replaced
by appropriate nutrient medium for animal cell culture.

As in the case of bacteriophage assay, the viral sample is serially

diluted and aliquots of appropriate dilutions are spread on monolayers of
host culture cells growing on a solid support e.g. in a petridish. The virions
are allowed to get attached to the host cells for an hour or so, and then the
monolayer is covered with a soft agar or some other gelling medium to
prevent free horizontal diffusion of the viral particles.

The infectious progeny particles released by lysis of the infected cells

remain more or less localized to produce foci or plaques. The plaques can
be counted to determine the infectivity titre of the virus sample. Development
of visible plaques may require from 1 to 2 days or even several weeks
depending on the virus. For facilitating detection and counting of plaques, it
may be necessary to stain the cell layer with a dye like neutral red which
stains only the living cells, or a stain like trypan blue which stains only the
dead cells. The accuracy of plaque assay depends on the number of
plaques counted.

If too many plaques develop on a plate, some of these tend to fuse.

As a result, the count becomes lower than what it should be. Many viruses
produce sharp plaques, while others produce plaques with a diffuse margin.
Again some viruses produce large plaques and others small ones.
Depending on the nature of the virus to be assayed, the dilution is to be
accordingly determined to get reliable results. In case of bacteriophages, the
number of plaques in plates is proportional to the concentration of the virus

150
i.e. the relationship between plaque number and the viral concentration is
linear.

Another method of assay of animal viruses which was used

previously and now practically abandoned and replaced by the plaque assay
is the pock assay. In this method the appropriately diluted viral sample is
inoculated into the epithelial layer of the chrioallantoic membrane (CAM) of a
chick embryo in an embryonated chicken egg. Characteristic infection
lesions, called pocks appear after an incubation period of 36 to 72 hr. The
pocks are opaque areas — usually white — and can be located on the
transparent CAM.

The plaque method may also be suitably modified for enumerating

plant viruses. For this purpose, a known volume of a properly diluted sample
of the plant virus is applied on the leaf surface of a susceptible host plant
after the leaf has been mechanically injured by mildly rubbing the surface
with an abrasive like carborandum.

After incubation for several days necrotic lesions appear on the leaf.
From the number of lesions per unit area, the dilution factor of the applied
viral sample and the inoculum volume, the concentration (titre) of the virus
can be calculated.

Cultivation of Viruses

As obligate parasites, viruses cannot be grown in inanimate artificial

media like bacteria or fungi. They can multiply only in living cells.

(i) Embryonated Egg

One of the earliest — but still a common method of cultivating animal

viruses — is the use of fertilized chicken eggs or embryonated eggs
containing a young (6-12 days old) embryo. Different animal viruses can
multiply in different parts of such eggs.

151
 Fig. 3.4 The different parts of an embryonated chicken egg

Of the different parts of an embryonated egg, the allantoic cavity and

the chorioallantoic membrane (CAM) are used for cultivating most animal
viruses, though the yolk sac and the embryo are also used for certain
viruses. The CAM is inoculated for growing pox viruses. The lesions (pocks)
are visible grayish white spots. Mumps virus grows preferentially in the
allantoic cavity. Influenza virus and rabies virus also grow in the allantoic
cavity. For inoculation, the egg surface is sterilized with a surface sterilizing
agent. A hole is drilled through the shell and the shell membrane and the
viral suspension is introduced into the proper part through the needle of a
hypodermic syringe. The hole is then sealed with paraffin and the inoculated
egg is incubated for 5 to 12 days.

(ii) Animal Cell Cultures:

The development of in vitro methods of culturing animal cells has

greatly improved propagation of animal viruses. Cultured animal cells have
provided quantitative techniques for studying animal viruses comparable to
those applied for studying bacterial viruses. Cultures of different origin can
be used for cultivating specific viruses, because just as all viruses cannot
infect and multiply in all host organisms, so also all viruses cannot be
propagated in all cell cultures.

Cultured animal cells differ in many ways from cultures of

microorganisms. One of the most important differences is that cell cultures

152
produced from normal tissues do not generally survive on repeated transfer.
After some time they do not further divide and die. Such cell cultures derived
from host tissues are called primary cultures. Primary cultures derived from
embryonic tissues are able to continue growth for a longer time than cell
cultures originating from adult tissues. For establishing a primary culture a
small piece of tissue of the animal is treated with trypsin to separate the
cells. Trypsin is removed by centrifugation and the cells are suspended. The
suspension is placed in a glass or plastic container together with a liquid
medium, like Eagle’s medium. On incubation, the cells attach to the surface
of the container and divide mitotically to produce daughter cells which
spread as a single-layer thick continuous confluent growth covering the
surface of the container (monolayer).

Primary cell cultures in monolayers can be inoculated with animal

viruses resulting in the formation of plaques. From the plaques, the viruses
can be collected and purified. Primary cultures can be prepared from various
tissues of different animals. The commonly used primary cultures are the
Rhesus monkey kidney cell culture, chick embryo fibrolast cell culture,
human amnion cell culture etc.

One of the limitations of the primary cell cultures is that they are
relatively short lived and cannot be indefinitely maintained in subcultures,
like those of microorganisms. The individual cells lose the capacity to divide
after several cell generations. Most human cell cultures lose dividing
capacity after about 50 duplications. Sometimes, it so happens that a clone
derived from a normal cell of a primary culture acquires the ability to grow
indefinitely. Such a clone having an unlimited dividing capacity gives rise to a
cell line. A cell line derived from a primary culture has a greater longevity
than the mother primary culture, but is not truly immortal.

During repeated transfer of a cell line, a clone of transformed cells

may originate spontaneously. These cells are cancer cells and are able to
produce cancer when introduced into the proper animal. Such transformed
cells constitute the permanent cell lines. Permanent cell lines can be
generated directly from cultures of malignant tissues of hosts, or they can be

153
produced infecting normal cell cultures with oncogenic viruses. The cells of
such permanent cell lines differ in morphology, cell orientation and
chromosomal make up from the cells of primary cultures and of the cell lines
derived from them. Different permanent cell lines have been generated from
different tissues and different sources e.g. human cervix, liver, amnion,
monkey kidney, hamster kidney, mouse connective tissue etc. one of the
best known permanent human cell line is the HeLa cells derived from the
cervix cancer of an Afro-American woman named Henrietta Lacks.

(iii) Bacteriophages

Cultivation of bacteriophages presents no difficulty, as they multiply

in growing host bacteria either in broth or on solidified medium. When an
inoculum containing a lytic phage is added to an actively growing liquid
bacterial culture, the turbidity falls and the culture becomes almost clear due
to lysis of the cells. By removing the cell debris by centrifugation, a rich crop
of the bacteriophage can be obtained.

Similarly, when a bacterial culture growing in a petridish is inoculated

with a phage suspension, the bacteria may be completely lysed, except a
few phage resistant colonies. If the number of bacteria far exceeds the
number of phages in the inoculum, then individual plaques are formed.
When a temperate bacteriophage Is inoculated into a liquid culture of the
appropriate host bacterium, the turbidity decreases transiently and then
increases. In agar cultures, the temperate phages produce turbid plaques.
Turbid plaques result because a small proportion of the host bacteria
undergo the lytic cycle, while the rest enters into lysogenic relationship.

(iv) Plant Viruses

Plant viruses are generally propagated by direct inoculation of

susceptible host plants. Viral inoculum is introduced into the leaf cells by
causing artificial mechanical injury, such as rubbing with an abrasive. The
viral particles enter through the disrupted cell walls into the cytoplasm of leaf
cells where they multiply. Some viruses, like TMV, may multiply in such great
numbers that the progeny TMV particles may account for as much as 10% of

154
the dry weight of the infected tobacco leaves. By disruption of the plant cells
and by differential centrifugation TMV can be isolated in mass.

In more recent times, some success has been obtained in cultivating

plant viruses in plant protoplast cultures. For example, protoplasts prepared
from tobacco leaf mesophyll cells can be infected with TMV. Some plant
viruses, like the Rhabdovirus causing necrotic yellow disease of lettuce, can
multiply both in the plant host as well as in the insect vector. Such viruses
can be grown in the vector cell culture. Lettuce necrotic yellow virus has
been successfully grown in monolayers of cell cultures derived from leaf
hopper giving an yield of about 10,000 virions per cell.

LET US SUM UP
Microbes - bacteria, archaea, fungi, algae, protozoa and viruses
have been around for at least 3,500 million years and were the only life
forms on Earth for most of that time. As the Earth cooled, liquid water formed
and the first microbial life appeared. The conditions on Earth in the beginning
were very hostile so the first microbes probably resembled the archaea, as
they were able to live in the extreme environments such as the high
temperature found on the cooling planet. Microbes affect every aspect of life
on earth. They have an amazing variety of shapes and sizes and can exist in
a wide range of habitats from hot springs to the icy wastes of Antarctica and
inside the bodies of animals and plants.

CHECK YOUR PROGRESS

1. Datura mosic virus transmitted through -----------

2. Nematode --------- transmit virus

GLOSSARY

1. Aphides - Aphids are the most notorious and important groups

of plant vector

155
SUGGESTED READINGS

1. Virology Principles and Application by John Carter

ANSWERS TO CHECK YOUR PROGRESS

1. Pollen

2. Trichodorus

MODEL QUESTIONS
1. Give a detailed account on Virus Transmission

156
UNIT 13

LIFE CYCLE OF VIRUS

STRUCTURE

Overview

Learning Objectives
13.1 The Lytic Cycle

13.2 The Lysogenic Cycle

13.3 Life Cycle of viruses with Animal hosts

Let us Sum up
Check your Progress
Glossary
Suggested Readings
Answers to check your progress
Model questions
OVERVIEW
All viruses depend on cells for reproduction and metabolic processes.
By themselves, viruses do not encode for all of the enzymes necessary for
viral replication. But within a host cell, a virus can commandeer cellular
machinery to produce more viral particles. Bacteriophages replicate only in
the cytoplasm, since prokaryotic cells do not have a nucleus or organelles. In
eukaryotic cells, most DNA viruses can replicate inside the nucleus, with an
exception observed in the large DNA viruses, such as the poxviruses, that
can replicate in the cytoplasm. RNA viruses that infect animal cells often
replicate in the cytoplasm.

LEARNING OBJECTIVES
To know the lifecycle of virus

157
 The life cycle of bacteriophages has been a good model for
understanding how viruses affect the cells they infect, since similar
processes have been observed for eukaryotic viruses, which can cause
immediate death of the cell or establish a latent or chronic infection. Virulent
phages typically lead to the death of the cell through cell lysis. Temperate
phages, on the other hand, can become part of a host chromosome and are
replicated with the cell genome until such time as they are induced to make
newly assembled viruses, or progeny viruses.

13.1 THE LYTIC CYCLE

During the lytic cycle of virulent phage, the bacteriophage takes

over the cell, reproduces new phages, and destroys the cell. T-seven phage
is a good example of a well-characterized class of virulent phages. There are
five stages in the bacteriophage lytic cycle. Attachment is the first stage in
the infection process in which the phage interacts with specific bacterial
surface receptors (e.g., lipopolysaccharides and OmpC protein on host
surfaces). Most phages have a narrow host range and may infect one
species of bacteria or one strain within a species. This unique recognition
can be exploited for targeted treatment of bacterial infection by phage
therapy or for phage typing to identify unique bacterial subspecies or strains.
The second stage of infection is entry or penetration. This occurs through
contraction of the tail sheath, which acts like a hypodermic needle to inject
the viral genome through the cell wall and membrane. The phage head and
remaining components remain outside the bacteria.

The third stage of infection is biosynthesis of new viral components.

After entering the host cell, the virus synthesizes virus-encoded
endonucleases to degrade the bacterial chromosome. It then hijacks the
host cell to replicate, transcribe, and translate the necessary viral
components (capsomeres, sheath, base plates, tail fibers, and viral
enzymes) for the assembly of new viruses. Polymerase genes are usually
expressed early in the cycle, while capsid and tail proteins are expressed
later. During the maturation phase, new virions are created. To liberate free
phages, the bacterial cell wall is disrupted by phage proteins such as holin or

158
lysozyme. The final stage is release. Mature viruses burst out of the host cell
in a process called lysis and the progeny viruses are liberated into the
environment to infect new cells.

13.2 THE LYSOGENIC CYCLE

In a lysogenic cycle, the phage genome also enters the cell through
attachment and penetration. A prime example of a phage with this type of life
cycle is the lambda phage. During the lysogenic cycle, instead of killing the
host, the phage genome integrates into the bacterial chromosome and
becomes part of the host. The integrated phage genome is called
a prophage. A bacterial host with a prophage is called a lysogen. The
process in which a bacterium is infected by a temperate phage is
called lysogeny. It is typical of temperate phages to be latent or inactive
within the cell. As the bacterium replicates its chromosome, it also replicates
the phage’s DNA and passes it on to new daughter cells during reproduction.
The presence of the phage may alter the phenotype of the bacterium, since
it can bring in extra genes (e.g., toxin genes that can increase bacterial
virulence). This change in the host phenotype is called lysogenic
conversion or phage conversion. Some bacteria, such as Vibrio
cholerae and Clostridium botulinum, are less virulent in the absence of the
prophage. The phages infecting these bacteria carry the toxin genes in their
genome and enhance the virulence of the host when the toxin genes are
expressed. In the case of V. cholera, phage encoded toxin can cause severe
diarrhea; in C. botulinum, the toxin can cause paralysis. During lysogeny, the
prophage will persist in the host chromosome until induction, which results
in the excision of the viral genome from the host chromosome. After
induction has occurred the temperate phage can proceed through a lytic
cycle and then undergo lysogeny in a newly infected cell.

Transduction

Transduction occurs when a bacteriophage transfers bacterial DNA

from one bacterium to another during sequential infections. There are two
types of transduction: generalized and specialized transduction. During the

159
lytic cycle of viral replication, the virus hijacks the host cell, degrades the
host chromosome, and makes more viral genomes. As it assembles and
packages DNA into the phage head, packaging occasionally makes a
mistake. Instead of packaging viral DNA, it takes a random piece of host
DNA and inserts it into the capsid. Once released, this virion will then inject
the former host’s DNA into a newly infected host. The asexual transfer of
genetic information can allow for DNA recombination to occur, thus providing
the new host with new genes (e.g., an antibiotic-resistance gene, or a sugar-
metabolizing gene).

Generalized transduction occurs when a random piece of bacterial

chromosomal DNA is transferred by the phage during the lytic
cycle. Specialized transduction occurs at the end of the lysogenic cycle,
when the prophage is excised and the bacteriophage enters the lytic cycle.
Since the phage is integrated into the host genome, the prophage can
replicate as part of the host. However, some conditions (e.g., ultraviolet light
exposure or chemical exposure) stimulate the prophage to undergo
induction, causing the phage to excise from the genome, enter the lytic
cycle, and produce new phages to leave host cells. During the process of
excision from the host chromosome, a phage may occasionally remove
some bacterial DNA near the site of viral integration. The phage and host
DNA from one end or both ends of the integration site are packaged within
the capsid and are transferred to the new, infected host. Since the DNA
transferred by the phage is not randomly packaged but is instead a specific
piece of DNA near the site of integration, this mechanism of gene transfer is
referred to as specialized transduction. The DNA can then recombine with
host chromosome, giving the latter new characteristics. Transduction seems
to play an important role in the evolutionary process of bacteria, giving them
a mechanism for asexual exchange of genetic information.

13.3 LIFE CYCLE OF VIRUSES WITH ANIMAL HOSTS

Lytic animal viruses follow similar infection stages to bacteriophages:

attachment, penetration, biosynthesis, maturation, and release. However,
the mechanisms of penetration, nucleic-acid biosynthesis, and release differ

160
between bacterial and animal viruses. After binding to host receptors, animal
viruses enter through endocytosis (engulfment by the host cell) or
through membrane fusion (viral envelope with the host cell membrane).
Many viruses are host specific, meaning they only infect a certain type of
host; and most viruses only infect certain types of cells within tissues. This
specificity is called a tissue tropism. Examples of this are demonstrated by
the poliovirus, which exhibits tropism for the tissues of the brain and spinal
cord, or the influenza virus, which has a primary tropism for the respiratory
tract.

13.4 Life Cycle of Viruses with Plant Hosts

Plant viruses are more similar to animal viruses than they are to
bacteriophages. Plant viruses may be enveloped or non-enveloped. Like
many animal viruses, plant viruses can have either a DNA or RNA genome
and be single stranded or double stranded. However, most plant viruses do
not have a DNA genome; the majority have a +ssRNA genome, which acts
like messenger RNA (mRNA). Only a minority of plant viruses have other
types of genomes. Plant viruses may have a narrow or broad host range. For
example, the citrus tristeza virus infects only a few plants of
the Citrus genus, whereas the cucumber mosaic virus infects thousands of
plants of various plant families. Most plant viruses are transmitted by contact
between plants, or by fungi, nematodes, insects, or other arthropods that act
as mechanical vectors.

LET US SUM UP
All viruses depend on cells for reproduction and metabolic processes.
By themselves, viruses do not encode for all of the enzymes necessary for
viral replication. But within a host cell, a virus can commandeer cellular
machinery to produce more viral particles. Bacteriophages replicate only in
the cytoplasm, since prokaryotic cells do not have a nucleus or organelles.

CHECK YOUR PROGRESS

161
 1. Give an example for Lytic cycle -------------------
2. Lysogenic virus virus ------------------

GLOSSARY

1. Endocytosis - engulfment by the host cell

SUGGESTED READINGS
1. Virology by Jay A levy et al

ANSWERS TO CHECK YOUR PROGRESS

1. T7 Phage
2. Lambda ohage

MODEL QUESTIONS
1. Write detailed notes on Life cycle of virus

162
UNIT 14

IMPORTANCE OF VIRUS AND

SPECIAL STUDY

STRUCTURE

Overview

Learning Objectives
14.1 Importance of Virus

14.2 Special Study

Let us Sum up
Check your Progress
Glossary
Suggested Readings
Answers to check your progress
Model questions

OVERVIEW
From the nuisance of the common cold to the debilitating symptoms
of AIDS, it’s rare to hear a positive story about a virus. But, as it turns out,
with the proper manipulations, viruses can do a lot more than cause disease.
In fact, scientists use genetically modified viruses to visualize the
connections between cells and treat disease.

LEARNING OBJECTIVES
To Understand the importance of virus

14.1 IMPORTANCE OF VIRUS

Vaccines The most direct approach to using viruses to prevent

cancers is simply that of vaccination against viruses that are associated with

163
cancer. Vaccines for hepatitis B virus (Hepadnaviridae; associated with
hepatocellular carcinoma) and human papillomavirus (Papillomaviridae;
associated with cervical cancer) are already available and are in widespread
use. Both use selected proteins of the virus. Although many studies have
been carried out, these are the only vaccines approved to date by the US
Food and Drug Administration (FDA) for the prevention of cancer. Of course,
few forms of cancer involve identifi ed viruses.

Virotherapy It is also possible to use the cell-killing effects of viruses

directly, rather than relying on the immune system. A range of viruses have
been used in efforts to produce targeted killing of cancerous cells, and the
approach as a whole is known as virotherapy. Some of the earliest evidence
for virotherapeutic approaches resulted from the observation of beneficial
effects in cancer patients associated with naturally occurring virus infections,
apparently due to the induction of innate immune responses. More recent
studies have shown that virus infection of cancer cells may enhance both
innate and adaptive immune responses. In the latter case, responses to both
viral and cancer cell antigens may be associated with beneficial effects.

Bacteriophages Bacteriophages are highly specific viruses that can

target, infect, and (if correctly selected) destroy pathogenic bacteria.
Antibacterial activity was fi rst observed in the waters of the Ganges and
Jumna rivers by Ernest Hankin in 1896. The causative agent was discovered
independently in 1915 by Frederick Twort and in 1917 by Felix d’Herelle. It
was d’Herelle who named them bacteriophages (devourers of bacteria) and
who then expanded on his initial fi nding to establish many of the techniques
that form the basis of modern virology. Bacteriophages are believed to be
the most numerous type of viruses accounting for the majority of the 1031
viruses present on Earth. They can be found at high concentrations in water,
with over 108 per milliliter being recorded from some sources. More than
90% of characterized bacteriophages are classifi ed in the order
Caudovirales. These are the tailed bacteriophages, with a large double-
stranded DNA genome in the range of 33,000 to 170,000 bp or even larger.

164
Other families of bacteriophages also exist, with a range of morphologies,
genome types, and genome sizes

14.2 SPECIAL STUDY

Bacteriophage

A bacteriophage, or phage for short, is a virus that infects bacteria.

Like other types of viruses, bacteriophages vary a lot in their shape and
genetic material. Phage genomes can consist of either DNA or RNA, and
can contain as few as four genes or as many as several hundred.

Structure

Bacteriophage come in many different sizes and shapes. The basic

structural features of bacteriophages are illustrated in Figure, which depicts
the phage called T4.

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.

Head of 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.

165
 Fig.3.1: Bacteriophage

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.

Life Cycle of Bacteriophage

166
 Bacteriophages are viruses that infect bacteria . Bacteriophages may
have a lytic cycle or a lysogenic cycle, and a few viruses are capable of
carrying out both. When infection of a cell by a bacteriophage results in the
production of new virions, the infection is said to be productive.

Lytic Cycle

With lytic phages, bacterial cells are broken open (lysed) and
destroyed after immediate replication of the virion. As soon as the cell is
destroyed, the phage progeny can find new hosts to infect. An example of a
lytic bacteriophage is T4, which infects E. coli found in the human intestinal
tract. Lytic phages are more suitable for phage therapy. Some lytic phages
undergo a phenomenon known as lysis inhibition, where completed phage
progeny will not immediately lyse out of the cell if extracellular phage
concentrations are high.

Lysogenic Cycle

In contrast, the lysogenic cycle does not result in immediate lysing of

the host cell. Those phages able to undergo lysogeny are known as
temperate phages. Their viral genome will integrate with host DNA and
replicate along with it fairly harmlessly, or may even become established as
a plasmid. The virus remains dormant until host conditions deteriorate,
perhaps due to depletion of nutrients; then, the endogenous phages (known
as prophages) become active. At this point they initiate the reproductive
cycle, resulting in lysis of the host cell. As the lysogenic cycle allows the host
cell to continue to survive and reproduce, the virus is reproduced in all of the
cell’s offspring. An example of a bacteriophage known to follow the lysogenic
cycle and the lytic cycle is the phage lambda of E. coli.

Latency Period

Viruses that infect plant or animal cells may also undergo infections
where they are not producing virions for long periods. An example is the
animal herpes viruses, including herpes simplex viruses, which cause oral
and genital herpes in humans. In a process called latency, these viruses can
exist in nervous tissue for long periods of time without producing new virions,

167
only to leave latency periodically and cause lesions in the skin where the
virus replicates. Even though there are similarities between lysogeny and
latency, the term lysogenic cycle is usually reserved to describe
bacteriophages.

Fig.14.1 Lytic Cycle

Cauliflower Mosaic Virus (CaMV)

Cauliflower mosaic virus (CaMV) is a type member of the

caulimoviruses which falls under the family Caulimoviridae in the Group VII
dsDNA-RT viruses. The CaMV are the only plant viruses that have dsDNA
genome. Due to the presence of dsDNA, the caulimoviruses are exploited as
vector in genetic engineering of plants. The CaMV consists of open circular
dsDNA as genetic material with single strand discontinuity like hepadna
Virus. The DNA is linear in situ but gets circularized after extraction. CaMV
shows icosahedral symmetry of the capsid with a 50 nm diameter. It consists
of more than one protein shell.

In theiytoplasm of infected cauliflower leaves, CaMV forms characteristic X

bodies which are rounded structure

168
Symptoms of CaMV

From the cell walls of infected leaves, there arise finger like
processes. It has also been observed that mitochondria and nuclei become
abnormal in the infected cells of host leaves.

Structure of CaMV

Virions occur in the cytoplasm, and in some cases in the nucleus.

Cytoplasmic virions are associated with electron-dense proteinaceous
inclusion bodies (for caulimoviruses, the product of ORF6). The product of
0RF2 also forms inclusion bodies which are electron translucent. Inclusion
bodies can be seen by light microscopy as well as by electron microscopy.
The virion CaMV shows an icosahedral symmetry with a diameter of 52 nm
which is made up of 420 capsid protein (CP) subunits arranged with a
triangulation T = 7.

The protein subunits surround a solvent-filled central cavity. Each

virion consists of a circular dsDNA molecule of about 8.0 kb which is
interrupted by site-specific discontinuities.

It results from its replication by reverse transcription. The single

stranded nicks in the viral DNA are repaired after entering the host. It results
in formation of a supercoiled molecule that binds to histone proteins.
Transcription of this DNA results into a full length 35S RNA and a sub-
genomic 19S RNA molecule, which are terminally redundant.

169
Fig. 14.2 CaMV A- Under EM; B and C Diagramatic

Genome of CaMV

The genome is a mono-partite, open circular dsDNA of about 8000

base pairs with one discontinuity in one strand, and one or more
discontinuity in the other strand.

Fig. 14.4 Open circular ds DNA of CaMV

Strands displacement causes gaps in the genome, which are associated

with the replication of the virus. The promoter of the 35S RNA is very strong.
It is used in plant transformation. The 35S RNA is complex and contains one
leader sequence (600 nt long). This leader is followed 6 to 8 tightly arranged
longer ORFs that encode all the viral proteins. There are six major coding
regions (I, III, IV, V, VI, VI11) and 2 minor coding regions (II. VII) in the
genome. The minor coding regions act as the store house of the non-
essential genes. The coding regions of ORFs and their function are given in
Table 16.4. The mechanism of expression of these proteins is very special.

170
 The ORF VI protein (encoded by the 19S RNA) controls translation
re-initiation of major open reading frames oh the polycistronic 35S RNA,
which results in release of virions. After making association with polysomes
and eukaryotic initiation factor eIF3, TAV starts its function.

Table. 14.1 Coding region and Function

Replication of CaMV

CaMV replicates by reverse transcription in cytoplasm/nucleus. Virion

of CaMV interacts with a cellular receptor of the host and enters inside the
cell. The viral dsDNA genome is transported to the nucleus where the host
RNA polymerase II carries out its transcription. Then translation of 35S RNA
and 19S RNA takes place where viral proteins are produced thereafter,
reverse transcription of 35S RNA occurs to produce new dsDNA genomes in
cytoplasm. Capsid protein encapsulates the viral genome. The new viral
particles get targeted to inclusion body and are released outside.

HIV

Human Immunodeficiency Virus (HIV) is a complex RNA virus of the

genus Lentivirus within the Retroviridae family. HIV is an approximately 100
nm icosahedral structure with 72 external spikes that are formed by the two
major envelope glycoproteins gp120 and [Link] major types of the AIDS
virus, HIV- 1 and HIV-2, have been identified. The major serological

171
differences reside in the surface protein gp120. HIV-1 and HIV-2 are
further separated into subtypes or ‘clades’ due to the marked variability in
the V3 (variable region) of the gp120 protein.

HIV encodes 3 structural genes and 6 regulatory genes.

1. Group Specific Antigen (Gag): Group antigens

2. Envelope (Env): Envelope

3. Polymerase (Pol): Polymerase

Fig.14.5 Structure of HIV

The gag Protein

gag is one of the three “main” genes found in all retroviruses (along
with envand pol). It contains around 1500 nucleotides, and encodes four
separate proteins which form the building blocks for the viral core:

Capsid protein, CA,

Matrix protein, MA

Nucleocapsid protein

172
The most significant role of the gag gene is therefore to encode important
proteins which will make up the viral core.

The pol protein

The gene pol is one of the main retroviral genes. It encodes four
proteins, of which the most important is Reverse Transcriptase. Reverse
Transcriptase performs a job which is unique to retroviruses, in that it copies
the virus’ RNA genome into DNA. (Since most organisms and viruses keep
their genes in DNA form in the first place, they have no need to perform this
task.) The copying of the HIV genome into DNA form is one of the key stages
of the HIV life-cycle. The other three products of pol are these:

Protease – which processes proteins made from HIV’s genome so that they
can become part of new fully-functioning HIV particles

RNAse H – which breaks down the retroviral genome following infection of a

cell

Integrase – which integrates the DNA copy of HIV’s genome into the
host DNA

The env protein

The “env” gene in HIV encodes a single protein, gp160. (When

gp160 is synthesised in the cell, cellular enzymes add complex carbohydrates
and turn it from a protein into a glycoprotein – hence the name “gp160″ rather
than “p160″.) gp160 travels to the cell surface, where cellular enzymes again
attack it, this time chopping into two pieces – gp120, and gp41. If and when
new virus particles bud off from the host cell, these two pieces lie on opposite
sides of the virus membrane. gp120 sits on the outside of the virus particle,
forming the virus’s spikes, while gp41 sits just on the inside of the membrane
– each gp41 being anchored to a gp120 through the membrane.

Assembly

Assembly involves the collection of all the components necessary for

the formation of the mature virion at a particular site in the cell. During
assembly, the basic structure of the virus particle is formed. The site of

173
assembly depends on the site of replication within the cell and on the
mechanism by which the virus is eventually released from the cell. As with
the early stages of replication, it is not always possible to identify the
assembly, maturation & release of virus particles as distinct and separate
phases.

Maturation

Maturation is the stage of the replication-cycle at which the virus

becomes infectious. Maturation usually involves structural changes in the
virus particle which may result from specific cleavages of capsid proteins to
form the mature products or conformational changes in proteins during
assembly. Such events frequently lead to substantial structural changes in
the capsid which may be detectable by criteria such as differences in the
antigenicity of incomplete & mature virus particles or the condensation of
nucleoproteins with the virus genome. Virus proteases are frequently
involved in maturation, although cellular enzymes or a mixture of virus &
cellular enzymes are used in some cases.

Release

Apart from plant viruses which have evolved particular strategies to

overcome the structure of plant cell walls, all other viruses escape the cell by
one of two mechanisms: For lytic viruses (most non-enveloped viruses),
release is a simple process - the infected cell breaks open & releases the
virus. Enveloped viruses acquire their lipid membrane as the virus buds out
of the cell through the cell membrane or into an intracellular vesicle prior to
subsequent release. Virion envelope proteins are picked up during this
process as the virus particle is extruded - this process is known as budding.
Preassembled capsomers are joined to form empty capsids (procapsid) The
assembly of capsomers to form the procapsid is often accompanied by
extensive reorganization, which is revealed by changes in serological
specificity and isoelectric point. eg. picornaviruses and adenoviruses.

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 Fig.3.8.6: Release of Virus

TMV

The tobacco mosaic virus affects all dicotyledonous plants of which

most important are tobacco and tomato. But it does not affect any
monocotyledonous plants. The tobacco mosaic virus affects photosynthetic
tissue of the host leading to distortion, blistering and necrosis. It also causes
dwarfing of affected plants. It is one of the most damaging viruses of plants,
causes enormous loss of tobacco crop by reducing yield and quality.

Structure of Tobacco Mosaic Virus (TMV)

TMV is a simple rod-shaped helical virus consisting of centrally

located single- stranded RNA (5.6%) enveloped by a protein coat (94.4%).
The rod is considered to be 3,000 Å in length and about 180 Å in diameter.
The protein coat is technically called ‘capsid’. R. Franklin estimated 2,130
sub-units, namely, capsomeres in a complete helical rod and 49 capsomeres
on every three turns of the helix; thus there would be about 130 turns per rod
of TMV. The diameter of RNA helix is about 80 Å and the RNA molecule lies
about 50 Å inward from the outer-most surface of the rod. The central core of
the rod is about 40 Å in diameter. Each capsomere is a grape like structure
containing about 158 amino acids and having a molecular weight of 17,000
dalton as determined by Knight.

175
 Fig.3.8.7: Strucutre of TMV

Life-Cycle (Replication) of Tobacco Mosaic Virus (TMV)

Plant viruses like TMV penetrate and enter the host cells in toto and
their replication completes within such infected host cells. Inside the host
cell, the protein coat dissociates and viral nucleic acid becomes free in the
cell cytoplasm. Although the sites for different steps of the viral multiplication
and formation of new viruses have not yet been determined with absolute
certainty, the studies suggest ha alter becoming free in the cell cytoplasm
the viral-RNA moves into the nucleus (possibly into the nucleolus).

The viral-RNA first induces the formation of specific enzymes called

‘RNA polymerases’ the single-stranded viral-RNA synthesizes an additional
RNA strand called replicative RNA.

176
 Fig.14.8:Replication of TMV

This RNA strand is complementary to the viral genome and serves as

‘template’ for producing new RNA single strands which is the copies of the
parental viral-RNA. The new viral-RNAs are released from the nucleus into
die cytoplasm and serve as messenger-RNAs (mRNAs). Each mRNA, in
cooperation with ribosomes and t-RNA of the host cell directs the synthesis
of protein subunits. After the desired protein sub-units (capsomeres) have
been produced, the new viral nucleic acid is considered to organize the
protein subunit around it resulting in the formation of complete virus particle,
the virion. No ‘lysis’ of the host cell, as seen in case of virulent
bacteriophages, takes place. The host ells remain alive and viruses move
from one cell to the other causing systemic infection. When transmitted by
some means the viruses infect other healthy plants.

4.3.5 Wound tumor virus

Wound tumor virus is an invertebrate and plant virus belonging to

the genus Phytoreovirus and the family Reoviridae. The virus is a Type III
virus under the Baltimore classification system; that is it has a double-
stranded RNA genome. This genome is approximately 25,000 base pairs

177
long and organised into twelve segments. All the viral replication occurs in
the cytoplasm. The virus is 22% RNA by weight, the other 78% being
structural proteins. Structurally, the virus is constructed from 7 different
structural proteins. The capsid has icosahedral symmetry, is non-enveloped
and around 70 nm in diameter. There is an inner-shell with a diameter of
around 50 nm. More than 50 species of plants are potential hosts for Wound
tumor virus. It was first reported in Melilotus officinalis. The virus causes
tumors to form on the plant at the stem and roots – with the root tumors
being more severe. The virus is spread by an insect vector – the Leaf
hopper family, notably 'Agallia constricta'. Since viral replication occurs
relatively independently of cellular processes, the virus also replicates in the
insect vector.

Structure

Phytoreovirus

VIRION

Fig. 3.8.9 Wound virus structure

Non enveloped, icosahedral, non-turreted virion with a double capsid

structure, about 70 nm in diameter. The outer capsid has a T=13 icosahedral

178
symmetry, the inner capsid a T=2* icosahedral symmetry. Each double-
layered capsid consists of 260 trimers of the major outer capsid P8 protein
and 60 dimers of the inner capsid P3 protein.

Gene Expression

The dsRNA genome is never completely uncoated, to prevent

activation of antiviral state by the cell in response to dsRNA. The viral
polymerase synthesizes a capped mRNA from each dsRNA segment. This
capped mRNA is translocated to the cell cytoplasm where it is translated.
RDV expresses an additional protein by leaky scanning

LET US SUM UP
Microbes affect every aspect of life on earth. They have an amazing
variety of shapes and sizes and can exist in a wide range of habitats from
hot springs to the icy wastes of Antarctica and inside the bodies of animals
and plants. The importance of virus and the special example has been
discussed in this unit.

CHECK YOUR PROGRESS

1. The Stucture of HIV is ----------
2. TMV does not affect --------

GLOSSARY
1. Protease - which processes proteins made from HIV’s genome
so that they can become part of new fully-functioning HIV
particles

SUGGESTED READINGS
1. Text book of Virology by Dutta Somnath

ANSWERS TO CHECK YOUR PROGRESS

1. Icosohedron
2. Monocots

MODEL QUESTIONS
1. Write notes on Tobacco Mosic Virus

179
 Block IV

IMMUNOLOGY

Unit 15: Introduction to Immunity

Unit 16: Immune System

Unit 17 The Immune Responseand Immunoglobulins

Unit 18: Antigen –Antibody Reaction

180
UNIT 15

INTRODUCTION TO IMMUNITY
STRUCTURE

Overview

Learning Objectives
15.1 Innate Immunity
15.2 Aquired Immunity
15.3 Immune Response
Let us Sum up
Check your Progress
Glossary
Suggested Readings
Answers to check your progress
Model questions

OVERVIEW
Immunology is the study of the immune system, which defends the
body against foreign intrusion by pathogens. The immune system
distinguishes self from nonself and eliminates potentially harmful nonself
molecules and cells from the body. The immune system also has the
capacity to recognize and destroy abnormal cells that derive from host
tissues. Any molecule capable of being recognized by the immune system is
considered an antigen.

LEARNING OBJECTIVES
To understand the Immunity of a body

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15.1 INNATE IMMUNITY

Innate immunity is non-specific, and represents the inherent

capability of the organism to offer resistance against diseases. It consists of
defensive barriers.

First line of defense

The skin is the largest organ in the human body, constituting about
15% of the adult body weight. The skin provides mechanical barrier to
prevent the entry of microorganisms and viruses. The acidic (pH 3-5)
environment on the skin surface inhibits the growth of certain
microorganisms. Further, the sweat contains an enzyme lysozyme that can
destroy bacterial cell wall.

Second line of defense

Despite the physical barriers, the microorganisms do enter the body.

The body defends itself and eliminates the invading organisms by non-
specific mechanisms such as sneezing and secretions of the mucus. In
addition, the body also tries to kill the pathogens by phagocytosis (involving
macrophages and complement system). The inflammatory response and
fever response of the body also form a part of innate immunity.

Innate or Natural or Nonspecific Immunity (L. innatus = inborn

Innate immunity is inherited by the organism from the parents and

protects it from birth throughout life. For example humans have innate
immunity against distemper, a fatal disease of dogs. As its name nonspecific
suggests that it lacks specific responses to specific invaders. Innate
immunity or nonspecific immunity is well done by providing different barriers
to the entry of the foreign agents into our body. Innate immunity consists of
four types of barriers— physical, physiological, cellular and cytokine barriers.

1. Physical Barriers

They are mechanical barriers to many microbial pathogens. These are of two
types. Skin and mucous membrane.

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(a) Skin

The skin is physical barrier of body. Its outer tough layer, the stratum
corneum prevents the entry of bacteria and viruses.

(b) Mucous Membranes

Mucus secreted by mucous membrane traps the microorganisms and

immobilises them. Microorganisms and dust particles can enter the
respiratory tract with air during breathing which are trapped in the mucus.
The cilia sweep the mucus loaded with microorganisms and dust particles
into the pharynx (throat). From the pharynx it is thrown out or swallowed for
elimination with the faeces.

2. Physiological Barriers

The skin and mucous membranes secrete certain chemicals which

dispose off the pathogens from the body. Body temperature, pH of the body
fluids and various body secretions prevent growth of many disease causing
microorganisms. Some of the important examples of physiological barriers
are as follows:

(a) Acid of the stomach kills most ingested microorganisms,

(b) Bile does not allow growth of microorganisms,

(c) Cerumen (ear wax) traps dust particles, kills bacteria and repels insects,

(d) Lysozyme is present in tissue fluids and in almost all secretions except in
cerebrospinal fluid, sweat and urine. Lysozyme is in good quantity in tears
from eyes. Lysozyme attacks bacteria and dissolves their cell walls.
Lysoenzyme is also found in saliva,

(e) Nasal Hair. They filter out microbes and dust in nose,

(f) Urine. It washes microbes from urethra,

(g) Vaginal Secretions. It is slightly acidic which discourages bacterial growth

and flush microbes out of vagina,

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(h) Sebum (sweat). It forms a protective acid film over the skin surface that
inhibits growth of many microbes.

3. Cellular Barriers

These are certain white blood corpuscles (leucocytes), macrophages,

natural killer cells, complement system, inflammation, fever, antimicrobial
substances, etc.

(i) Certain Leucocytes

Neutrophils and monocytes are major phagocytic leucocytes.

(a) Polymorpho-nuclear Leucocytes (PMNL- neutrophils)

As they have multilobed nucleus they are normally called

polymorphonuclear leucocytes (PMNL-neu- trophils). Neutrophils are short
lived and are highly motile phagocytic killers. Neutrophils are formed from
stem cells in the bone marrow. Neutrophils are the most numerous of all
leucocytes. They die after a few days and must therefore, be constantly
replaced. Neutrophils constitute about 40% to 75% of the blood leucocytes in
humans.

(b) Monocytes

They are the largest of all types of leucocytes and somewhat

amoeboid in shape. They have clear cytoplasm (without cytoplasmic
granules). The nucleus is bean-shaped. Monocytes constitute about 2-10%
of the blood leucocytes. They are motile and phagocytic in nature and engulf
bacteria and cellular debris. Their life span is about 10 to 20 hours.
Generally they change into macrophages after entering tissue spaces.

(ii) Macrophages

Monocytes circulate in the bloodstream for about 8 hours, during

which time they enlarge and then migrate into the tissues and differentiate
into specific tissue macrophages. Macrophages are long lived and are highly
motile phagocytic. Macrophages contain more cell organelles especially
lysosomes. Macrophages are of two types, (a) Some take up residence in

184
particular tissues becoming fixed macroph- ages and (b) whereas other
remain motile and are called wandering macrophages. Wandering
macrophages move by amoeboid movement throughout the tissues. Fixed
macrophages serve different functions in different tissues and are named to
reflect their tissue location. Some examples are given below

i. Pulmonary alveolar macrophages in the lung

ii. Histiocytes in connective tissues

iii. Kupffer cells in the liver

iv. Glomerular Mesangial cells in the kidney

v. Microglial cells in the brain

vi. Osteoclasts in bone

(iii) Natural Killer Cells (NK Cells)

Besides the phagocytes, there are natural killer cells in the body
which are a type of lymphocytes and are present in the spleen, lymph nodes
and red bone marrow. NK cells do not have antigen receptors like T cells
and В cells. NK cells cause cellular destruction in at least two ways:

(a) NK cells produce perforins which are chemicals that when inserted into
the plasma membrane of a microbe make so weak that cytolysis (breakdown
of cells particularly their outer membrane) occurs and creates pores in the
plasma membrane of the target cells. These pores allow entry of water into
the target cells, which then swell and burst. Cellular remains are eaten by
phagocytes.

(b) Another function of NK cells is apoptosis which means natural cell death.
It occurs naturally as part of the normal development, maintenance and
renewal of cells, tissues and organs.

Thus functions of NK cells are to destroy target cells by cytolysis and

apoptosis. NK cells constitute 5%-10% of the peripheral blood lymphocytes
in humans.

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(iv) Complement

Complement is a group of 20 proteins, many of which are enzyme

precursors and are produced by the liver. These proteins are present in the
serum of the blood (the fluid portion of the blood excluding cells and clotting
factors) and on plasma membranes. They are found circulating in the blood
plasma and within tissues throughout the body. They were named
complement by Ehrlich because they complement the actions of other
components of the immune system (e.g., action of antibody on antigen) in
the fight against infection. Jules Bordet is the discoverer of complement.

Fig. 15.1 Complement proteins

Complement proteins create pores in the plasma membrane of the microbes.

Water enters the microbes. The latter burst and die. The proteins of
complement system destroy microbes by (i) cytolysis (ii) inflammation and
(iii) phagocytosis. These proteins also prevent excessive damage of the host
tissues.

(v) Inflammation

Inflammation is a defensive response of the body to tissue damage.

The conditions that may produce inflammation are pathogens, abrasions
(scraping off) chemical irritations, distortion or disturbances of cells, and

186
extreme temperatures. The signs and symptoms of inflammation are
redness, pain, heat and swelling.

Inflammation can also cause the loss of function in the injured area,
depending on the site and extent of the injury. Inflammation is an attempt to
dispose of microbes, toxins, or foreign material at the site of injury to prevent
their spread to other tissues, and to prepare the site for tissue repair. Thus, it
helps restore tissue homeostasis.

Broken mast cells release histamine. Histamine causes dilation of

capillaries and small blood vessels. As a result more blood flows to that area
making it red and warm and fluid (plasma) takes out into the tissue spaces
causing its swelling. This reaction of the body is called inflammatory
response.

(vi) Fever

Fever may be brought about by toxins produced by pathogens and a

protein called endogenous pyrogen (fever producing substance), released by
macrophages. When enough pyrogens reach the brain, the body’s
thermostat is reset to a higher temperature, allowing the temperature of the
entire body to rise. Mild fever strengthens the defence mechanism by
activating the phagocytes and by inhibiting the growth of microbes. A very
high temperature may prove dangerous. It must be quickly brought down by
giving antipyretics.

4. Cytokine Barriers

Cytokines (Chemical messengers of immune cells) are low molecular

weight proteins that stimulate or inhibit the differentiation, proliferation or
function of immune cells. They are involved in the cell to cell communication.
Kinds of cytokines include interleukins produced by leucocytes, lymphocytes
produced by lymphocytes, tumour necrosis factor and interferon’s (IFNs).
Interferon’s protect against viral infection of cells.

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15.2 ACQUIRED IMMUNITY

The immune system represents the third and most potent defense
mechanism of the body. Acquired (adaptive or specific) immunity is capable
of specifically recognizing and eliminating the invading microorganisms and
foreign molecules (antigens).

In contrast to innate immunity, the acquired immunity displays four

distinct characteristics

i. Antigen specificity

ii. Recognition diversity

iii. Immunological memory

iv. Discrimination between self and non-self.

The body possess tremendous capability to specifically identify

various antigens (antigen is a foreign substance, usually a protein or a
carbohydrate that elicits immune response). Exposure to an antigen leads to
the development of immunological memory. As a result, a second encounter
of the body to the same antigen results in a heightened state of immune
response. The immune system recognizes and responds to foreign antigens
as it is capable of distinguishing self and non-self. Autoimmune diseases are
caused due to a failure to discriminate self and non-self-antigens.

(B) Acquired Immunity (= Adaptive or Specific Immunity)

The immunity that an individual acquires after the birth is called

acquired or adaptive or specific immunity. It is specific and mediated by
antibodies or lymphocytes or both which make the antigen harmless. It not
only relieves the victim of the infectious disease but also prevents its further
attack in future. The memory cells formed by В cells and T cells are the
basis of acquired immunity. Thus acquired immunity consists of specialized
В and T lymphocytes and Antibodies.

Characteristics of Acquired Immunity

(i) Specificity

188
It is the ability to differentiate between various foreign molecules (foreign
antigens).

(ii) Diversity

It can recognise a vast variety of foreign molecules (foreign antigens).

(iii) Discrimination between Self and Non-self

It can recognise and respond to foreign molecules (non-self) and can

avoid response to those molecules that are present within the body (self) of
the animal.

(iv) Memory

When the immune system encounters a specific foreign agent, (e.g.,

a microbe) for the first time, it generates immune response and eliminates
the invader. This is called first encounter. The immune system retains the
memory of the first encounter. As a result, a second encounter occurs more
quickly and abundantly than the first encounter.

The cells of the immune system are derived from the pluripotent stem
cells in the bone marrow. Pluripotent means a cell that can differentiate into
many different types of tissue cells. The pluripotent stem cells can form
either myeloid stem cells or lymphoid stem cells. Myeloid stem cells give rise
to monocytes, macrophages and granulocytes (neutrophils eosinophil’s, and
basophils). RBCs and blood platelets (lymphoid stem cells) form В lym-
phocytes (B cells), T lymphocytes (T-cells) and natural killer (NK) cells.

Components of Acquired Immunity

Acquired immunity has two components: humeral immunity or

Antibody mediated immune system (AMIS) and cellular immunity or cell
mediated immune system (CMIS).

189
 Fig. 15.2 Development of B and T lymphocytes

I. Antibody Mediated Immune System (AMIS) or Humoral Immunity

It consists of antibodies (specialised proteins produced in the body in

response to antigen) that circulate in the body fluids like blood plasma and
lymph. The word ‘humor’ pertains to fluid. В lymphocytes (B cells) produce
antibodies that regulate humoral immunity. The T-lymphocytes themselves
do not secrete anti-bodies but help В lymphocytes produce them. Certain
cells of the bone marrow produce В lymphocytes and mature there. Since В
lymphocytes produce antibodies, therefore, this immunity is called antibody
mediated or humoral immunity. Humoral immunity or antibody-mediated

190
immune system (AMIS) provides defence against most extracellular bacterial
pathogens and viruses that infect through the respiratory and intestinal tract.

Formation of Plasma В cells and Memory В cells

When antibodies on В cell’s surface bind antigens (any substances

that cause antibodies formation) the В cell is activated and divides,
producing a clone (descendants of a single cell) of daughter В cells. These
clones give rise to plasma В cells and memory В cells. This phenomenon is
called clonal selection.

(a) Plasma В Cells (Effector В cells)

Some of the activated В cells enlarge, divide and differentiate into a

clone of plasma cells. Although plasma cells live for only a few days, they
secrete enormous amounts of antibody during this period.

(b) Memory В Cells

Some activated В cells do not differentiate into plasma cells but

rather remain as memory cells (Primed cells). They have a longer life span.
The memory cells remain dormant until activated once again by a new
quantity of the same antigen.

Role of AMIS

The AMIS protects the body from (i) viruses (ii) some bacteria and
(iii) toxins that enter the body fluids like blood and lymph.

II. Cell-Mediated Immune System (CMIS) or Т-Cell Immunity

A healthy person has about a trillion lymphocytes. Lymphocytes are

of two types: T lymphocytes or T cells and В lymphocytes or В cells. As we
know both types of lymphocytes and other cells of the immune system are
produced in the bone marrow. The process of production of cells of immune
system in the bone marrow is called haematopoiesis.

Because T lymphocytes (T cells) mature in the thymus, this immunity is also

called T- cell immunity.

The T-cells play two important functions—effector and regulatory.

191
The effector function includes cytolysis (destruction of cells by immune
processes) of cells infected with microbes and tumour cells and lymphokine
production. The regulatory functions are either to increase or to suppress
other lymphocytes and accessory cells.

Types of T-cells and their Functions

1. Helper T cells (TH)

TH cells are most numerous of the T cells. They help in the functions
of immune system. They produce a growth factor that stimulates В-cell
proliferation and differentiation and also stimulates antibody production by
plasma cells; enhance activity of cytotoxic T cells.

2. Cytotoxic T cells (Tc) or Killer cells

These cells are capable of killing microorganisms and even some of

the body’s own cells directly hence they are called killer cells. The antigen
receptors on the surfaces of the cytotoxic cells cause specific binding with
antigens present on the surface of foreign cell.

Cell after binding, the cytotoxic T cell secretes hole-forming proteins,

called perforins, that punch large round holes in the membrane of the foreign
cell. Then fluid flows quickly into the cell from the interstinal space. In
addition, the cytotoxic T cell releases cytotoxic substances directly into the
foreign cell. Almost immediately, the foreign cell becomes greatly swollen
and it usually dissolves shortly thereafter.

Thus they destroy body cells infected by viruses and attack and kill bacteria,
fungi, parasites and cancer cells.

192
 Fig. 15.3 Cytotoxic T-cell

3. Memory T Cells (Primed Cells)

These cells are also formed by T-lymphocytes as a result of

exposure to antigen and remain in the lymphatic tissue (e.g., spleen, lymph
nodes). They recognize original invading antigens even years after the first
encounter. These cells keep ready to attack as soon as the same pathogens
infect the body again. They proliferate and differentiate into cytotoxic T cells,
helper T cells, suppressor T cells, and additional memory cells.

4. Suppressor Cells (Regulatory T cells (TR))

These cells are capable of suppressing the functions of cytotoxic and

helper T cells. They also inhibit the immune system from attacking the
body’s own cells. It is believed that suppressor cells regulate the activities of
the other cells. For this reason, the suppressor cells are classified as
regulatory T cells.

Natural Killer (NK) Cells

NK cells attack and destroy target cells, participate in antibody

dependent cell mediated cytotoxicity. They can also attack parasites which
are much larger than bacteria.

193
Types of Acquired Immunity

Acquired (= Adaptive) Immunity is of two types: active immunity and passive

immunity.

1. Active Immunity

In this immunity person’s own cells produce antibodies in response to

infection or vaccination. It is slow and takes time in the formation of
antibodies. It is long lasting and is harmless. Active immunity may be natural
or artificial.

(a) A person who has recovered from an attack of small pox or measles or
mumps develops natural active immunity.

(b) Artificial active immunity is the resistance induced by vaccines. Examples

of vaccines are as follows: Bacterial vaccines, (a) Live- BCG vaccine for
tuberculosis, (b) Killed vaccines- TAB vaccine for enteric fever. Viral
vaccines, (a) Live – sabin vaccine for poliomyelitis, MMR vaccine for
measles, mumps, rubella, (b) Killed vaccines- salk vaccine for poliomyelitis,
neural and non-neural vaccines for rabies. Bacterial products. Toxoids for
Diphtheria and Tetanus.

2. Passive Immunity

When ready-made antibodies are directly injected into a person to

protect the body against foreign agents, it is called passive immunity. It
provides immediate relief. It is not long lasting. It may create problems.
Passive immunity may be natural or artificial.

(a) Natural passive immunity is the resistance passively transferred from the
mother to the foetus through placenta. IgG antibodies can cross placental
barrier to reach the foetus. After birth, immunoglobulin’s are passed to the
new-born through the breast milk. Human colostrum (mother’s first milk) is
rich in IgA antibodies. Mother’s milk contains antibodies which protect the
infant properly by the age of three months.

(b) Artificial passive immunity is the resistance passively transferred to a

recipient by administration of antibodies. This is done by administration of

194
hyper-immune sera of man or animals. Serum (pi. sera) contains antibodies.
For example, anti-tetanus serum (ATS) is prepared in horses by active
immunisation of horses with tetanus toxoid, bleeding them and separating
the serum. ATS is used for passive immunisation against tetanus. Similarly
anti-diphtheric serum (ADS) and anti-gas gangrene serum (AGS) are also
prepared.

15.3 IMMUNE RESPONSE

The immune response involves primary immune response and

secondary immune response.

(a) The primary immune response

After an initial contact with an antigen, no antibodies are present for a

period of several days. Then, a slow rise in the antibody titer o(arbitrary
units) occurs, first IgM and then IgG followed by a gradual decline in
antibody titer. This is called the primary immune response.

(b) The secondary immune response

Memory cells may remain in the body for decades. Every new
encounter with the same antigen results in a rapid proliferation of memory
cells. This is also called “booster response”. The antibody titer after
subsequent encounters is far greater than during a primary response and
consists mainly of IgG antibodies. This accelerated, more intense response
is called the secondary immune response. Antibodies produced during a
secondary response have an even higher affinity for the antigen.

A person who had been suffering from diseases like measles, small
pox or chicken pox becomes immune to subsequent attacks of these
diseases. It includes spleen, lymph nodes, tonsils, Peyer’s patches of small
intestine and appendix.

The increased power and duration of the secondary immune

response explain why immunization (method of providing immunity
artificially, it is called vaccination) is usually accomplished by injecting
antigen in multiple doses.

195
LET US SUM UP
Immunology deals with the study of immunity and immune systems of
vertebrates. Immunity (immunis literally means exempt/free from burden)
broadly involves the resistance shown, and protection offered by the host
organism against the infectious diseases. The immune system consists of a
complex network of cells and molecules, and their interactions. It is
specifically designed to eliminate infectious organisms from the body. This is
possible since the organism is capable of distinguishing the self from non-
self, and eliminate non-self. Immunity is broadly divided into two types —
innate (non-specific) immunity and adaptive or acquired (specific) immunity.

CHECK YOUR PROGRESS

1. Innate immunity is -------
2. Plasma B cells otherwise called ----------------

GLOSSARY
1. Monocytes - They are the largest of all types of leucocytes and
somewhat amoeboid in shape.

SUGGESTED READINGS
1. Cellular and Molecular Immunology by Adil K Abbas

ANSWERS TO CHECK YOUR PROGRESS

1. non specific
2. Effector B cells

MODEL QUESTIONS
1. Write Detailed notes on Aquired Immunity

196
UNIT - 16

ORGANIZATION OF IMMUNE SYSTEM

STRUCTURE

Overview

Learning Objectives
16.1 Lymphoid Organs
16.2 Primary Lymphoid Organs
16.3 Secondary Lymphoid Organs
Let us Sum up
Check your Progress
Glossary
Suggested Readings
Answers to check your progress
Model questions

OVERVIEW
The immune system distinguishes self from nonself and eliminates
potentially harmful nonself molecules and cells from the body. The immune
system also has the capacity to recognize and destroy abnormal cells that
derive from host tissues. Any molecule capable of being recognized by the
immune system is considered an antigen (Ag).

LEARNING OBJECTIVES
To understand the organisation of Immune system

16.1 LYMPHOID ORGANS

The immune system consists of several organs distributed

throughout the body. These lymphoid organs are categorized as
primary and secondary.

197
 Fig. 16.1 Human Lymphatic system

16.2 PRIMARY LYMPHOID ORGANS

These organs provide appropriate micro- environment for the

development and maturation of antigen-sensitive lymphocytes (a type of
white blood cells). The thymus (situated above the heart) and bone marrow
are the central or primary lymphoid organs. T-lymphocyte maturation occurs
in the thymus while B-lymphocyte maturation takes place in the bone
marrow.

Primary or Central Lymphoid Organs

Immature lymphocytes generated in hematopoiesis, the process of

formation and development of blood cells, mature and become committed to
a particular antigenic specificity within the primary lymphoid organs, namely,
thymus, bursa of Fabricius (in birds) and bone marrow (in mammals). A
lymphocyte becomes immuno-competent, i.e., capable of mounting an
immune response only after it matures within a primary lymphoid organ.

198
1. Thymus

Thymus is a greyish, flat, bilobed lymphoid organ situated above the

heart and extending into the neck on the front and sites of trachea. It
develops from the epithelium of third and fourth pharyngeal pouches and, on
maturity, acts as the site of development and maturation of lymphocytes
named thymus-derived lymphocytes or T-lymphocytes or T-cells.

The thymus reaches peak activity in childhood and attains its largest
size at puberty. Thereafter, the thymus begins to atrophy without any
apparent effect on T-lymphocyte function and is extremely small in old age.

For convenience, the average weight of the thymus is 70 g in infants

and its age- dependent involution leaves the thymus with an average weight
of 3 g in the old age. This is probably due to the fact that T-lymphocytes are
very long-lived and can circulate in the resting state for long periods of time.

Each lobe of thymus is surrounded by a capsule and is divided into a

series of lobules, which are separated from each other by strands of
connective tissue called trabeculae. Each lobule is organized into two
compartments-outer and inner. The outer component is called cortex,
whereas the inner component is called medulla .

The cortex is densely packed with thymocytes, whereas the medulla

is sparsely populated with thymocytes. Thymocytes develop from
prothymocytes. The latter are produced in bone marrow, migrate through
blood stream, enter the cortex of the thymus, and act as thymocytes.
Thymocytes divide rapidly in the cortex and give rise to T-lymphocytes.

Of the T-lymphocytes produced in thymus only 5% leave the thymus

as viable cells. Though the reason for this apparent wasteful process is not
known, some believe that it is the elimination of T-lymphocyte clones that
react against self.

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 Fig. 16.2 C.S of Thymus

Both the cortex and the medulla of the thymus are criss-crossed by a
three dimensional network consisting of epithelial cells, dendritic cells, and
macrophages, which make up the framework of the organ and contribute to
the growth and maturation, of thymocytes.

Some epithelial cells of the outer cortex possess long membrane

extensions that surround as many as 50 thymocytes. These cells are called
nurse cells. Other epithelial cells of the cortex have long interconnecting
cytoplasmic extensions that form a network and have been found to interact
with many of the thymocytes when they traverse the cortex. The function of
the thymus is to generate T-lymphocytes and to confer immunological
competence on to them during their stay in the organ. T-lymphocytes so
educated in the thymus become capable of mounting cell-mediated immune
response against appropriate antigen. This is effected under the influence of
the thymic microenvironment and several hormones such as thymosin and
thymopietin produced by the epithelial cells of the thymus. The competent T-
lymphocytes immediately move from thymus to the secondary or peripheral
lymphoid organs.

2. Bursa of Fabricius

Bursa of Fabricius is a primary lymphoid organ in birds where stem

cells from yolk sac, foetal lever, and bone marrow mature, proliferate, and
differentiate into bursa-derived lymphocytes called B-lymphocytes or B-cells.
Bursa of Fabricius arises as a pouch from the dorsal part of cloaca (fluid gut)

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in birds, Bursa of Fabricius is sensitive to hormones: administration of
testosterone at the early embryostage completely prevents its formation
(hormonal bursectomy).

Surgical removal of bursa (bursectomy) from newly hatched chickens

destroys their subsequent ability to produce antibodies. The B-cells mature,
proliferate, and differentiate into bursa and then migrate from it and reach
outer or superficial cortex of the germinal follicles and medullary cords of
peripheral lymph nodes and lymphoid follicles of spleen where, following
appropriate antigenic stimulation, transform into plasma cells and secrete
antibodies. Like thymus, the bursal of Fabricius starts to shrink or atrophy at
puberty.

3. Bone Marrow

Bone marrow is the site of origin and development of B-lymphocytes

or B-cells (bone marrow derived lymphocytes) in mammals particularly in
humans and mice after birth. Before birth, the yolk sac, foetal lever, and total
bone marrow are the major sites of B-lymphocyte maturation. Bone marrow,
therefore, is the mammalian equivalent of the bursa of Fabricius in birds.

Development of B-lymphocytes (B-cells) begins with the

differentiation of lymphoid stem cells into the earliest distinctive progenitor B
cells (pro-B cell), which proliferate within the bone marrow filling the
extravascular spaces between large sinusoids in the shaft of a bone.
Proliferation and differentiation of pro-B cells into precursor B cells (pre-B
cells) requires the microenvironment provided by the bone marrow stromal
cells.

The stromal cells within the bone marrow

(1) Interact directly with the pro-B and pre-B cells and

(2) Secrete various cytokines that are required for development.

Bone marrow is not the site of origin and development of B-

lymphocytes (B-cells) in all mammals. In cattle and sheep, the fietal spleen is
the primary lymphoid tissue wherein the maturation, proliferation, and

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diversification of B-cells take place during early gestation. During later
gestation this function is performed by ideal Peyer’s patch, a patch of tissue
embedded in the wall of the intestine. In rabbit, gut-associated tissues (e.g..
appendix) act as primary lymphoid tissue for maturation, proliferation, and
diversification of B-cells.

Secondary or Peripheral Lymphoid Organs

As stated earlier, the lymphocytes mature, proliferate, and

differentiate in the primary or central lymphoid organs. These lymphocytes
migrate therefrom via circulation to the secondary or peripheral lymphoid
organs. Here they bind appropriate antigens and undergo further antigen-
dependent differentiation. Once in the secondary lymphoid organs, the
lymphocytes do not remain there but move from one lymphoid organ to
another through the blood and lymphatic’s. The passage of lymphocytes
facilitates the induction of an immune response. Lymph nodes and the
spleen are the most highly organized secondary or peripheral lymphoid
organs, whereas mucosa-associated lymphoid tissue (MALT) is the less
organized lymphoid tissue.

1. Lymph Nodes

Lymph nodes are small, encapsulated, bean-shaped structures

clustered at junctions of the lymphatic vessels which are distributed
throughout the body. Lymph nodes contain a reticular network packed with
lymphocytes, macrophages and dendritic cells, and filter out pathogenic
microorganisms and antigens from the lymph. As the lymph percolates
through a lymph node, any pathogen or antigen that is brought in with the
lymph is trapped by the phagocytic cells and dendritic cells.

A lymph node consists of three regions: the cortex, the paracortex,

and the medulla. Cortex is the outermost region and contains several
rounded aggregates of lymphocytes (mostly B-lymphocytes), macrophages,
and follicular dendritic cells arranged in primary follicles. Each follicle has a
pale-staining germinal centre surrounded by small dark-staining
lymphocytes. The deeper region lying beneath the cortex is the paracortex. It

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is the zone between the cortex and the medulla. Paracortex possesses large
number of T-lymphocytes and also contains inter-digitating dendritic cells
thought to have migrated from tissues to the lymph node. Because of the
presence of large number of T-lymphocytes in it. the Para-cortex is also
referred to as a thymus-dependent area in contrast to the cortex which is a
thymus-independent area. Medulla, the inner most region of lymph node, is
more sparsely populated with lymphoid-lineage cells. Of the lymphoid-
lineage cells present, many are plasma cells actively secreting antibody
molecules. Each lymph node has a number of lymph vessels called afferent
lymphatic vessels, which pierce the capsule of a lymph node at numerous
sites and empty lymph into the sub-capsular sinus. The lymph now
percolates slowly inward through the cortex, paracortex, and medulla,
allowing phagocytic cells and dendritic cells to trap pathogens and antigens
carried by the lymph. The lymph then is drained into a single large lymph
vessels called efferent lymphatic vessel that carries the lymph to the thoracic
duct, which empties into a large vein in the neck.

Fig. 16.3 Lymph node

2. Spleen

The spleen, which is about 5 inches long and 200 g in weight in

adults, is an ovoid encapsulated, and the largest secondary or peripheral
lymphoid organ. Spleen is specialized for trapping blood-borne antigens and
is present high in the left abdominal cavity and being encapsulated, its

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capsule extends a number of projections, called trabeculae, into the interior
resulting in the formation of compartments. These compartments are filled by
two types of tissues, the red pulp and white pulp, which are separated by a
diffuse marginal zone. The red pulp consists of a network of sinusoids
populated by large number of erythrocytes (red blood cells) and
macrophages and few lymphocytes.

In fact, red pulp is the region where old and defective erythrocytes
are destroyed and eliminated. The white pulp consist of the branches of the
splenic artery that make a periarteriolar lymphoid sheath (PALS) populated
heavily by T-lymphocytes. Periarteriolar lymphoid sheath (PALS) is attached
with primary lymphoid follicles that are rich in B-lymphocytes. The marginal
zone separating the red pulp from white pulp is populated by lymphocytes
and macrophages. When the blood-borne antigens enter the spleen the B-
and T-lymphocytes present in periarteriolar lymphoid sheath (PALS) are
initially activated. Here interdigitating dendritic cells capture antigen and
present it combined with class II MHC molecules (major histocompatibility
molecules) to TH cells (T helper cells). Once activated, these TH cells can
then activate B- lymphocytes (B-cells).

The activated B-lymphocytes, together with some TH cells then

migrate to primary follicles in the marginal zone. When the primary follicles
are challenged by antigen, they differentiate into characteristic secondary
follicles. The latter contain germinal centres (similar to those occurring in
lymph nodes) where rapidly dividing B-lymphocytes and plasma cells are
surrounded by dense clusters of concentrically arranged lymphocytes.

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 Fig. 16.4 Lymphoid organ

3. Mucosal-Associated Lymphoid Tissue (MALT)

The mucous membranes lining the alimentary, respiratory, and

genitourinary systems have a very large combined surface area (about 400
m2; nearly the size of a basketball court), which is constantly exposed to
numerous antigens and is the major site of entry for most pathogens.

These vulnerable membrane surfaces possess a group of organized

lymphoid tissues which defend it from pathogens and antigens. The group of
organized lymphoid tissues is known collectively as mucosal-associated
lymphoid tissue (MALT). There are several types of MALT; the most studied
one is the gut-associated lymphoid tissue (GALT) which includes tonsils,
Peyer’s patch, appendix, and loosely organised clusters of lymphoid cells in
the lamina propria of intestinal villi.

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 Mucosal-associated lymphoid, tissue (MALT) is functionally very
significant in immune system of the body because of the presence of large
number of antibody-producing plasma cells in it. The number of plasma cells
in MALT for exceeds that of the total of the number of plasma cells present
in spleen, lymph nodes, and bone marrow.

(i) Tonsils

There are three groups of tonsil present at three different locations:

palatine, lingual, and pharyngeal (adenoids). Palatine group of tonsil occur at
the sides of the back of the mouth; lingual in the basal region of the tongue;
and pharyngeal (adenoids) in the roof of the nasopharynx. All the aforesaid
tonsil groups are nodule-like and consist of a meshwork of reticular cells and
fibres interspersed with lymphocytes, macrophages, granulocytes, and mast
cells.

The B-lymphocytes are organised into follicles and germinal centres.

The germinal centres are surrounded by regions showing T-lymphocyte
activity. However, the tonsils protect against antigens that enter through the
nausal and oral epithelial routes.

Fig.16.4 Three types if tonsils

(ii) Peyer’s Patch

Peyer’s patches occur in the sub-mucosal layer present beneath the

lamina propria lying under the epithelial layer of intestinal villi. Each Peyer’s

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patch is a nodule of 30-40 lymphoid follicles. Like lymphoid follicles in other
sites, those that compose Peyer’s patches can develop into secondary
follicles with germinal centres.

Fig. 16.5 Peyer’s patch

(iii) Lamina Propria

Lamina propria occurs under the epithelial layer of intestinal villi. It is

populated with large number of plasma cells, macrophages, activated T
helper cells (activated TH cells) in loose clusters. More than 15,000 lymphoid
follicles have beer, reported within the lamina propria of a healthy child.

16.2 SECONDARY LYMPHOID ORGANS

These are the sites for the initiation of immune response, e.g. spleen,
tonsils, lymph nodes, appendix, Peyers patches in the gut. Secondary
lymphoid organs provide the microenvironment for interaction between
antigens and mature lymphocytes.

Cells of the Immune System

Two types of lymphocytes namely B-cells and T-cells are critical for
the immune system. In addition, several accessory cells and effector cells
also participate. Lymphocytes are one of the five kinds of white blood
cells or leukocytes), circulating in the blood. Although mature lymphocytes

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all look pretty much alike, they are extraordinarily diverse in their functions.
The most abundant lymphocytes are:

B lymphocytes (often simply called B cells) and

T lymphocytes (likewise called T cells).

B cells are produced in the bone marrow. The precursors of T cells are also
produced in the bone marrow but leave the bone marrow and mature in
the thymus (which accounts for their designation). Each B cell and T cell
is specific for a particular antigen. What this means is that each is able
to bind to a particular molecular structure. The specificity of binding resides
in a receptor for antigen:

 the B cell receptor (BCR) for antigen and

 the T cell receptor (TCR) respectively.

Both BCRs and TCRs share these properties:

 They are integral membrane proteins.

 They are present in thousands of identical copies exposed at the cell

surface.

 They are made before the cell ever encounters an antigen.

 They are encoded by genes assembled by the recombination of

segments of DNA.

 They have a unique binding site.

 This site binds to a portion of the antigen called an antigenic

determinant or epitope.

 The binding, like that between an enzyme and its substrate depends
on complementarity of the surface of the receptor and the surface of
the epitope.

 The binding occurs by non-covalent forces (again, like an enzyme

binding to its substrate).

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  Successful binding of the antigen receptor to the epitope, if
accompanied by additional signals, results in:

o stimulation of the cell to leave G0 and enter the cell cycle.

o Repeated mitosis leads to the development of a clone of cells

bearing the same antigen receptor; that is, a clone of cells of
the identical specificity.

B-lymphocytes

The site of development and maturation of B-cells occurs in bursa

fabricus in birds, and bone marrow in mammals. During the course of
immune response. B-cells mature into plasma cells and secrete antibodies
(immunoglobulin’s). The B-cells possess the capability to specifically
recognize each antigen and produce antibodies against it. B-lymphocytes
are intimately associated with humoral immunity. BCRs bind intact antigens
(like diphtheria toxoid, the protein introduced into your body in the DTP
vaccine). These may be soluble molecules present in the extracellular fluid;
intact molecules that the B cell plucks from the surface of antigen-presenting
cells like macrophages and dendritic cells. The bound antigen molecules are
engulfed into the B cell by receptor-mediated endocytosis. The antigen is
digested into fragments which are then displayed at the cell surface nestled
inside a class II histocompatibility molecule. Helper T cells specific for this
structure (i.e., with complementary TCRs) bind the B cell and
secrete lymphokines that: stimulate the B cell to enter the cell cycle and
develop, by repeated mitosis, into a clone of cells with identical BCRs;
switch from synthesizing their BCRs as integral membrane proteins to a
soluble version; differentiate into plasma cells that secrete these soluble
BCRs, which we now call antibodies.

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 Fig. 16.6 B and T - cell

T-lymphocytes

The maturation of T-cells occurs in the thymus, hence the name. The
T-cells can identify viruses and microorganisms from the antigens displayed
on their surfaces.

There are at least four different types of T-cells

i. Inducer T-cells that mediate the development of T-cells in the thymus.

ii. Cytotoxic T-cells (Tc), capable of recognizing and killing the infected or
abnormal cells.

iii. Helper T-cells (TH) that initiate immune responses.

iv. Suppressor T-cells mediate the suppression of immune response.

T-lymphocytes are responsible for the cell- mediated immunity.

The surface of each T cell also displays thousands of identical T cell

receptors (TCRs).

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There are two types of T cells that differ in their TCR: alpha/beta (αβ) T
cells. Their TCR is a heterodimer of an alpha chain with a beta chain. Each
chain has a variable (V) region and a constant (C) region. The V regions
each contain 3 hypervariable regions that make up the antigen-binding
site. gamma/delta (γδ) T cells. Their TCR is also a heterodimer of a gamma
chain paired with a delta chain. The discussion that follows now concerns
alpha/beta T cells. Gamma/delta T cells, which are less well understood, are
discussed at the end. The TCR (of alpha/beta T cells) binds a bimolecular
complex displayed at the surface of some other cell called an antigen-
presenting cell (APC). This complex consists of:

 a fragment of an antigen lying within the groove of a

 histocompatibility molecule

The complex has been compared to a "hot dog in a bun". Most of the T cells
in the body belong to one of two subsets. These are distinguished by the
presence on their surface of one or the other of
two glycoproteins designated:

CD4

CD8

Which of these molecules is present determines what types of cells the T cell
can bind to.

CD8+ T cells bind epitopes that are part of class I histocompatibility

molecules. Almost all the cells of the body express class I molecules.

CD4+ T cells bind epitopes that are part of class II histocompatibility

molecules. Only specialized antigen-presenting cells express class II
molecules. These include:

o dendritic cells

o phagocytic cells like macrophages

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LET US SUM UP
The immune system is complex and pervasive. There are numerous
cell types that either circulate throughout the body or reside in a particular
tissue. Each cell type plays a unique role, with different ways of recognizing
problems, communicating with other cells, and performing their functions. By
understanding all the details behind this network, researchers may optimize
immune responses to confront specific issues, ranging from infections to
cancer.

CHECK YOUR PROGRESS

1. Thymus situated in -----
2. Bursa of fabricius is lymphoid organ in -----------------

GLOSSARY
1. Lymph node - Lymph nodes are small, encapsulated,
bean-shaped structures clustered at junctions of the
lymphatic vessels

SUGGESTED READINGS
1. Immunology by Janis Kuby

ANSWERS TO CHECK YOUR PROGRESS

1. above the heart
2. birds

MODEL QUESTIONS
1. Write detailed account on immune response

212
UNIT - 17

THE IMMUNE RESPONSE AND

IMMUNOGLOBUIN
STRUCTURE

Overview

Learning Objectives
17.1 Immune Response
17.2 Cytokines
17.3 Immunoglobulins
Let us Sum up
Check your Progress
Glossary
Suggested Readings
Answers to check your progress
Model questions

OVERVIEW
Immunoglobulin (also called gamma globulin or immune globulin) is a
substance made from human blood plasma. The plasma, processed from
donated human blood, contains antibodies that protect the body against
diseases. When you are given an immunoglobulin, your body uses
antibodies from other people's blood plasma to help prevent illness. And
even though immunoglobulins are obtained from blood, they are purified so
that they can't pass on diseases to the person who receives them..

LEARNING OBJECTIVES
To understand the role of immunoglobulins

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17.1 IMMUNE RESPONSE

The immune response refers to the series of reactions carried out by

the immune system in the body against the foreign invader. When an
infection takes place or when an antigen enters the body, it is trapped by the
macrophages in lymphoid organs. The phagocytic cells which are guarding
the body by constant patrolling engulf and digest the foreign substance.
However, the partially digested antigen (i.e. processed antigen) with
antigenic epitopes attaches to lymphocytes. T-helper cells (TH) play a key
role the immune response. This is brought out through the participation of
antigen presenting cell (APC), usually a macrophage. Receptors of TH cell
bind to class II MHC-antigen complex displayed on the surface of APC. APC
secretes interleukin-l, which activates the TH cell. This activated TH cell
actively grows and divides to produce clones of TH cells.

Fig. 17.1 Helper T-Cells

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All the TH cells possess receptors that are specific for the MHC-antigen
complex. This facilitates triggering of immune response in an exponential
manner. The TH cells secrete interleukin-2 which promotes the proliferation
of cytotoxic T cells (TC cells) to attack the infected cells through cell-
mediated immunity. Further, interleukin-2 also activates B-cells to produce
immunoglobulin’s which perform humoral immunity.

17.2 CYTOKINES

Cytokines are a group of proteins that bring about communication

between different cell types involved in immunity. They are low molecular
weight glycoproteins and are produced by lymphoid and non-lymphoid cells
during the course of immune response. Cytokines may be regarded as
soluble messenger molecules of immune system.

They can act as short messengers between the cells or long range
messengers by circulating in the blood and affecting cells at far off sites. The
latter function is comparable to that of hormones. The term interleukin (IL) is
frequently used to represent cytokines. There are more than a dozen
interleukins (IL-I…… IL12)/ produced by different cells with wide range of
functions. The main function (directly or indirectly) of cytokines is to amplify
immune responses and inflammatory responses.

Therapeutic uses of cytokines

It is now possible to produce cytokines in vitro. Some of the cytokines

have potential applications in the practice of medicine. For instance, IL-2 is
used in cancer immunotherapy, and in the treatment of immunodeficiency
diseases. IL-2 induces the proliferation and differentiation of T-and B-cells,
besides increasing the cytotoxic capacity of natural killer cells. A group of
cytokines namely interferon’s can combat viral infection by inhibiting their
replication.

17.3 IMMUNOGLOBULIN

The humoral immunity is mediated by a special group of proteins

called immunoglobulin’s or antibodies, produced by B-lymphocytes.

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Structure of Immunoglobulins

All the immunoglobulin (Ig) molecules basically consist of two

identical heavy (H) chains (mol. wt. 53,000 to 75,000 each) and two identical
light (L) chains (mol. wt. 23,000 each) held together by disulfide linkages and
non-covalent interactions. Thus, immunoglobulin is a Y-shaped tetramer
(H2L2). Each heavy chain contains approximately 450 amino acids while
each light chain has 212 amino acids. The heavy chains of Ig are linked to
carbohydrates, hence immunoglobulin’s are glycoproteins.

Fig. 17.2 Diagramatic representation of IgG

Constant and variable regions

Each chain (L or H) of Ig has two regions (domains), namely the

constant and the variable. The amino terminal half of the light chain is the
variable region (VL) while the carboxy terminal half is the constant region
(CL). As regards heavy chain, approximately one-quarter of the amino
terminal region is variable (VH) while the remaining three-quarters is constant

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(CH1, CH2, CH3). The amino acid sequence (with its tertiary structure) of
variable regions of light and heavy chains is responsible for the specific
binding of immunoglobulin (antibody) with antigen.

Classes of Immunoglobulin

Humans have five classes of immunoglobulin’s— namely IgG, IgA,

IgM, IgD and IgE— containing the heavy chains ƴ, α, µ, δ and ɛ,
respectively. The type of heavy chain ultimately determines the class and the
function of a given Ig. Two types of light chains — namely kappa (k) and
lambda (λ) — are found in immunoglobulin’s. They differ in their structure in
CLregions. An immunoglobulin (of any class) contains two k or two λ light
chains and never a mixture. The occurrence of k chains is more common in
human immunoglobulin’s than λ chains.

The characteristics of the 5 classes of human immunoglobulin’s are given in

Table.

Immunoglobulin G (IgG)

IgG is the most abundant (75-80%) class of immunoglobulin’s. IgG is

composed of a single Y-shaped unit (monomer). It can traverse blood
vessels readily. IgG is the only immunoglobulin that can cross the placenta

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and transfer the mother’s immunity to the developing fetus. IgG triggers
foreign cell destruction mediated by complement system.

Immunoglobulin A (IgA)

IgA occurs as a single (monomer) or double unit (dimer) held

together by J chain. It is mostly found in the body secretions such as saliva,
tears, sweat, milk and the walls of intestine. IgA is the most predominant
antibody in the colostrum, the initial secretion from the mother’s breast after
a baby is born. The IgA molecules bind with bacterial antigens present on
the body (outer epithelial) surfaces and remove them. In this way, IgA
prevents the foreign substances from entering the body cells.

Immunoglobulin M (IgM)

IgM is the largest immunoglobulin composed of 5 Y-shaped units

(IgG type) held together by a J polypeptide chain. Thus IgM is a pentamer.
Due to its large size, IgM cannot traverse blood vessels; hence it is restricted
to the blood stream. IgM is the first antibody to be produced in response to
an antigen and is the most effective against invading microorganisms.

Immunoglobulin D (IgD)

IgD is composed of a single Y-shaped unit and is present in a low

concentration in the circulation. IgD molecules are present on the surface of
B cells. Their function, however, is not known for certain. Some workers
believe that IgD may function as B-cell receptor.

Immunoglobulin E (IgE)

IgE is a single Y-shaped monomer. It is normally present in minute

concentration in blood. IgE levels are elevated in individuals with allergies as
it is associated with the body’s allergic responses. The IgE molecules tightly
bind with mast cells which release histamine and cause allergy.

Synthesis of Immunoglobulin

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 There are millions of different antigens. It was a big puzzle for a long
time how an individual can produce so many antibodies to protect against
antigens. It is now recognized that a gene rearrangement involving a
combination of several genes is responsible for generating an extremely
large number of antibodies.

LET US SUM UP
The antibody immune response is highly complex and exceedingly
specific. The various immunoglobulin classes and subclasses (isotypes)
differ in their biological features, structure, target specificity and distribution.
Hence, the assessment of the immunoglobulin isotype can provide useful
insight into complex humoral immune response.

CHECK YOUR PROGRESS

1. Most abundant Immunoglobulin is -----------------
2. Cytokines madeup of -------

GLOSSARY
1. Macrophage – Antigen presenting cell

SUGGESTED READINGS
1. Essentials of Immunology by S. K. Gupta

ANSWERS TO CHECK YOUR PROGRESS

1. IgG
2. Proteins

MODEL QUESTIONS
1. Give a detailed account on Immunoglobulins

219
UNIT - 18

ANTIGEN-ANTIBODY REACTION
STRUCTURE

Overview

Learning Objectives
18.1 Antigen – Antibody reaction
18.2 Types of AG – AB reaction
Let us Sum up
Check your Progress
Glossary
Suggested Readings
Answers to check your progress
Model questions

OVERVIEW
The function of antibodies (Abs) involves specific binding to antigens
(Ags) and activation of other components of the immune system to fight
pathogens. The six hypervariable loops within the variable domains of Abs,
commonly termed complementarity determining regions (CDRs), are widely
assumed to be responsible for Ag recognition, while the constant domains
are believed to mediate effector activation.

LEARNING OBJECTIVES
To understan the antigen and antibody reaction

18.1 ANTIGEN - ANTIBODY REACTION

Antigen (Ag) antibody (Ab) reactions occur when an antigen

combines with a corresponding antibody to produce an immune complex.
Therefore, an antigen-antibody reaction is thus a bimolecular association
which is similar to an enzyme-substrate interaction but the only difference is

220
that antigen-antibody reaction does not lead to an irreversible chemical
interaction. The basis for antigen-antibody reactions are the non-covalent
interactions like hydrogen bonds, ionic bonds, van der Waal interactions,
hydrophobic interactions, etc. These interactions are individually weak,
therefore, a large number of such interactions work together in an antigen-
antibody reaction. The in vitro study of antigen antibody reactions is known
as serology. The principle for all diagnostic immunological tests is serological
reactions. The binding of an antibody with an antigen of the type that
stimulated the formation of the antibody, results in agglutination,
precipitation, complement fixation, greater susceptibility to ingestion and
destruction by phagocytes, or neutralization of an exotoxin. The main use of
antigen-antibody reactions is in the determination of blood groups for
transfusion, serological ascertainment of exposure to infectious agents, and
development of immunoassays for the quantification of various substances.

Schematically an Antigen-Antibody Reaction can be represented as:

Ag + Ab [Ag-Ab] → Aggregation → Precipitation/Agglutination/Neutralization

For diagnostic immunological tests, the serological tests must possess high
specificity and sensitivity. Specificity is the ability of an antibody to recognize
a single specific antigen. There is a high degree of specificity in antigen-
antibody reactions.

Antibodies can distinguish differences in:

i. Primary structure of an antigen,

ii. Isomeric forms of an antigen, and

iii. Secondary and tertiary structure of an antigen.

Therefore specificity implies that:

a. Antibody is specific for a single and specific antigen.

b. Antibody will not cross-react with other antigens.

c. It will not give false positive results.

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 Sensitivity means the lowest amount of antigen that can be detected.
If in a diagnostic test an antibody is capable of detecting a single antigen
molecule, then such a test possesses highest sensitivity. The amount of
antigen detected in a test is directly proportional to the amount of antibody
used. Enzyme Linked Immuno Sorbent Assays (ELISA) is the most sensitive
serological tests.

Fig. 18.1 Antibody structure

18.2 TYPES OF AG-AB REACTION

The following points highlight the eight main types of interaction

between antigen and antibody

1. Neutralization:

Certain antibodies called neutralizing antibodies react with antigen

and neutralize them so that they fail to attach on host cell surface.

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i. Toxin Neutralization:

Toxin neutralization is the process during which antibodies capable

neutralizing a toxin (neutralizing antibodies) or antiserum containing
neutralizing antibodies against a toxin inactivate the toxins produced by
bacterial pathogens. These neutralizing antibodies or antiserum containing
neutralizing antibodies are called antitoxins.

Once neutralized, the toxin-antibody complex is either unable to

attach to receptor sites on host target cells, unable to enter the cell, or it is
ingested by macrophages. However, bacterial pathogens causing tetanus,
diphtheria, etc. are some toxin-producing bacteria.

Fig. 18.2Toxin naturalization

Viral neutralization is the process during which specific antibodies

(IgG, IgM, and IgA) bind to viruses and inactivate them. Viral neutralization
prevents viral infection due to the inability of virus to bind to and enter its
target cell. However, fixation of the classical pathway, especially the
complement component C3b to the virus, also aids to viral neutralization
process.

Fig. 18.3 Viral naturalization

2. Immune Complex Formation

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 Antibodies possess at least two antigen-binding sites and most
antigens have at least two epitopes (antigenic determinants). The antibodies
cross-link antigens forming large aggregates of antibody and antigen
referred to as immune complexes, which are more readily phagocytized than
are free antigens.

Depending upon their physical properties, immune complex forming

antibodies are of two types: precipitins and agglutins. The precipitins react
with antigens that are soluble molecules and form immune complex large
enough to precipitate: this process is called precipitation (L. praecipitare = to
cast down) or precipitin reaction.

The agglutins, however, react with surface-bound antigens of

bacterial or other cells and form immune complex. This process is called
agglutination or agglutin reaction. Agglutination specifically involving red
blood cells is called hem-agglutination and is caused by antibodies called
hemagglutin.

Fig. 18.4 Immune Complex

3. Opsonization

Opsonization (G. opson = to prepare victim for) or opsonin-

dependent recognition is one of the two fundamental molecular mechanisms
used by phagocytic cells for the recognition of microbial pathogens and their
adherence on phagocyte’s plasma membrane so that the phagocytes can
ingest them easily.

4. Complement System

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 Complement fixation refers to the ability of antigen-antibody-complex
to bind complement so that the latter becomes “fixed” and “used up”. It is
operated by a system called the complement system which consists of over
30 soluble and cell-bound proteins and glycoproteins that interact in a highly
regulated cascade. This system involves in antigen-antibody interaction in
host to play its role in immune response.

5. Antibody-Dependent Cell-Medicated Cytotoxicity (ADCC)

Many cells that have cytotoxic potential express membrane receptors

for the Fc region of the antibody molecule. When antibody is specifically
bound to a target cell, the receptors of these cells can bind to the antibody
Fc region. Thus the cytotoxic cells bind to the target cells, and subsequently
cause lysis of the target cells.

Although these cytotoxic cells are nonspecific for the antigen, the specificity
of the antibody bound to the target cells directs the cytotoxic cells to specific
target cells. This type of cytotoxicity is referred to as antibody- dependent
cell-mediated cytotoxicity (ADCC).

The cells that can mediate ADCC are NK cells, macrophages,

monocytes, neutrophils, and eosinophils. ADCC to cells infected with the
measles virus can be observed in vitro by adding anti-measles antibody
together with macrophages to a culture of measles-infected cells. Similarly
cell-mediated killing of helminths, such as blood flukes, can be observed in
vitro by incubating larvae with antibody specific to the larvae together with
eosinophils. ADCC appears to involve a number of different cytotoxic
mechanisms, but it does not involve complement-mediated lysis. When
macrophages, neutrophils or eosinophils bind to a target cell, they become
more active metabolically. As a result, thelytic enzymes in their cytoplasmic
lysosomes (often referred to as granules) increase in quantity.

Release of these lytic enzymes at the site of the Fc-mediated contact

may result in damage to the target cell. In addition, activated monocytes,
macrophages, and NK cells have been shown to secrete tumor necrosis
factor (TNF), which may have a cytotoxic effect on the bound target cell.

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Since both NK cells and eosinophils contain perform in their cytoplasmic
granules, their target-cell killing also may involve perforin-mediated
membrane damage.

Fig. 18.5 Antibody dependent cell cytotoxicity

6. Hypersensitivity

Hypersensitivity is a condition of increased immune sensitivity in

which the body reacts to an antigen with an exaggerated immune response
that usually harms the individual by tissue damage. Hypersensitivity, also
termed an allergy, is manifested in the individual on second or subsequent
contact with an antigen and appears immediate or delayed.

7. Autoimmunity

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 Autoimmunity is a condition characterized by the presence of serum,
autoantibodies and self-reactive lymphocytes (T-cells). Autoimmunity (also
the autoimmune diseases) manifests sometimes when the body loses
immune tolerance (body’s condition to distinguish its own self-antigens from
foreign non-self-antigens and not mounting an immunogenic attack against
the former) and mounts an abnormal immune attack, either with antibodies
or T-cells, against a person’s own self-antigens.

8. Immuno-Deficiencies

Immunodeficiency refers to the inability of the immune system to

produce a normal complement of antibodies or immunologically sensitive T-
lymphocytes in response to self-antigens. Such inabilities of the immune
system can make the host more prone to infection and, as a result, the host
may become unable to protect itself from disease causing agents or from
malignant cells.

LET US SUM UP
Antibodies (Abs) have two distinct functions: one is to bind
specifically to their target antigens (Ags); the other is to elicit an immune
response against the bound Ag by recruiting other cells and molecules. The
association between an Ab and an Ag involves myriad of non-covalent
interactions between the epitope – the binding site on the Ag, and the
paratopes – the binding site on the Ab.

CHECK YOUR PROGRESS

1. Ag-Ab reaction Is a ------------
2. Antibody posses minimum ------------------ antigen biniding site

GLOSSARY
1. Opsonization – cells to prepare for ingetion

SUGGESTED READINGS
1. Immunobiology by Charles Janeway

227
ANSWERS TO CHECK YOUR PROGRESS
1. bimolecular association
2. 2

MODEL QUESTIONS
1. Wtite detailed account on Ag-Ab reaction

228
 Block V

PLANT PATHOLOGY

Unit 19: History Scope and Principles of plant infection

Unit 20: Principles of plant infection

Unit 21: Structural and Biochemical Defence Mechanism in Plants

Unit 22: Disease Resistance and incorporation of resistant genes

Unit 23: Methods for incorporation of resistant

229
UNIT - 19
HISTORY AND SCOPE OF PLANT
PATHOLOGY
STRUCTURE

Overview

Learning Objectives
19.1 Introduction to Plant Pathology

19.2 Concepts of Plant Disease

19.3 Symptoms of Plant Diseases

Let us Sum up
Check your Progress
Glossary
Suggested Readings
Answers to check your progress
Model questions

OVERVIEW
Plant Pathology is the study of the diseases and disorders of plants.
Disease can be defined as a harmful deviation from normal functioning of the
physiological processes caused by an infectious agent. In the case of plant
diseases, the causal agent maybe a fungus, virus, bacterium or a parasitic
flowering plant.

LEARNING OBJECTIVES
To know about the plant diseases and causal organisms.

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19.1 INTRODUCTION TO PLANT PATHOLOGY

Phytopathology (Phyton : plant) Greek - Pathos (suffering) + Logos

(study) = The study of the suffering plant. Plant pathology is that branch of
agricultural, botanical or biological sciences which deals with the study of:
cause of the disease Resulting losses and Control of plant diseases.

Stakman & Harrar (1957) defined disease as physiological disorder

or structural abnormality that is deleterious to the plant or its part or product,
that reduces the economic value of the plant e.g., wilt, potato blight, Loose
smut of wheat, karnal bunt of wheat.

A healthy plant Carries out its functions to the best of its genetic
potential Cells divide and differentiate as needed and specialized cells fulfill
dedicated tasks but Diseased Plant When the ability of cells of a plant or
plant part is compromised and Cells affected indicates type of physiological
function lost.

19.2 CONCEPTS OF PLANT DISEASE

Conditions Necessary for Disease In order for disease to
occur, three conditions must be met. First it is necessary to have a
susceptible host plant. Each species of plant can be infected by only some
pathogens. The plant must also be in a stage of development susceptible to
infection by the disease agent. The second requirement is the presence of
an active pathogen. If there is no pathogen present, there can be no
disease. Also, the pathogen must be in a stage of development conducive to
infecting or affecting the host plant. The third condition is an environment
suitable for the pathogen to cause disease of the plant. The interaction of
host, pathogen, and environment can some times be represented by a
triangle. The "disease triangle" cannot be constructed unless all 3 legs are
present simultaneously. Break any leg of the triangle, and there is no
disease. Disease control strategies can be based on breaking a leg of the
triangle.

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 Fig.19. 1: Disease triangle

Pathogen may Cause Disease in Plant by:

i) Weakening the host by continuously absorbing food from the host cells for
their own use.
ii) Killing or disturbing metabolism of host cells through toxins enzymes or
growth regulating substances, they secrete.
iii) Blocking the transportation of food, mineral, nutrients and water through
the conductive tissues.
iv) Consuming the contents of the host cells upon contact.
(i) Abiotic Factors:-
These are the resultants of deficiencies or excess of nutrients, light,
moisture, aeration, adverse soil conditions or atmospheric condition etc.
These are generally referred as disorders.
(ii) Mesobiotic Factors:-
The causal agent is neither living thing nor a non living thing. The
diseases caused by viroids and viruses are of this category.
(iii) Biotic Factors:-
This category includes diseases caused by living or cellular
organization. 1) Eukaryotes: - Fungi, Protozoa, Algae, Nematodes,
Parasites. 2) Prokaryotes : Mycoplasma, Rickettsia, Bacteria.

19.3 SYMPTOMS OF PLANT DISEASES

Evidence of disease shown by plant is called symptom. Symptoms
are seen on the plant either due to character and appearance of the visible
pathogen or its structure or organs or due to some effect upon or change in
the host plant.

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A) Symptoms due to visible pathogen

The pathogen is visible when they are in larger size or in sufficient mass,
such symptoms are as follows:

1) Mildew: - Pathogen is seen as a growth on the surface of the host.

Downy mildew and powdery mildew.

Fig.19.2: Comparison of Healthy and affected plant

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 Fig.19.3: Mildew & Rust disease

2) Rust: - Rusty symptoms are seen on the host epidermis Red, Green,
yellow and black rust.

3) Smuts: - Sooty or charcoal like powder appears on floral organs, smut

symptoms also found on stem, leaves and roots.

Fig.19.4: Smut of cereals

4) White blisters : Numerous white blister, like pustules are seen.

5) Scab: Crust like lesion on the diseased organs.

6) Sclerotia: A compact, often hard, mass of dormant fungus mycelium.

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 Fig.19.5 Sclerotia on Onion

7) Blotch: Superficial growth giving the fruit a blotched appearance.

8) Fruiting bodies: Relatively large spore bearing structures which are either
fleshy or woody.

9) Exudations: mass of bacteria oozes out to the surface of affected organ.

10) Tar spots: Raised, black coated fungus bodies with the appearance of a
flattened out drop of tar on leaves.

B) Symptoms due to some effect on host plant

1) Colour changes, Discoloration, Chromosis, and Chlorosis.

2) Overgrowth Galls, Curl, bladder, witches broom, hairy root

Fig.19.6: Gall

3) Dwarfing stunted growth

4) Necrosis Spots, strips, blight, damping off, scald, scorch, rot.

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 Fig.19.7: Necrosis

5) Anthracnose: ulcer like lesions on stems, leaves, pods.

6) Dieback: Drying of plant organ from the tip backwards.

7) Wilts : Drying or wilting of entire plant

Fig.19.8: Wilt of Banana

8) Miscellaneous symptoms. Change in habit, dropping of leaves, flowers,

fruits, destruction of organs etc.

LET US SUM UP
The major plant pathogens responsible for disease development in
plants are fungi, bacteria, viruses, and nematodes. The disease cycle
describes the interaction of the pathogen with the host

236
CHECK YOUR PROGRESS
1. Smut symptoms appear on ----------
2. Sclerotia is a disease common in -----

GLOSSARY
1. Symptoms - Evidence of disease shown by plant is called symptom.

SUGGESTED READINGS
1. Plant Pathology by R.S Mehrotra

ANSWERS TO CHECK YOUR PROGRESS

1. floral organs
2. onions

MODEL QUESTIONS
1. Write notes on Concepts of Plant Pathology

237
UNIT - 20
PRINCIPLES OF PLANT INFECTION
STRUCTURE

Overview

Learning Objectives
20.1 Inoculum Potential

20.2 Koch’s Pastulate

Let us Sum up
Check your Progress
Glossary
Suggested Readings
Answers to check your progress
Model questions

OVERVIEW
Losses in crop yields due to disease need to be reduced in order to
meet increasing global food demands associated with growth in the human
population. There is a well-recognized need to develop new environmentally
friendly control strategies to combat bacterial crop disease. Current control
measures involving the use of traditional chemicals or antibiotics are losing
their efficacy due to the natural development of bacterial resistance to these
agents. In addition, there is an increasing awareness that their use is
environmentally unfriendly. Bacteriophages, the viruses of bacteria, have
received increased research interest in recent years as a realistic
environmentally friendly means of controlling bacterial diseases.

LEARNING OBJECTIVES
To understand the principles of plant infection

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20.1 INOCULUM POTENTIAL
Garrett (1960) defined inoculum potential as the amount of energy
available for the fungus to infect the host at the site of infection. The
availability of nutrients affects the inoculum potential of the pathogen.

20.2 KOCH’S POSTULATES

The bacteria must be present in every case of the disease. The
bacteria must be isolated from the host with the disease and grown in pure
culture. The specific disease must be reproduced when a pure culture of the
bacteria is inoculated into a healthy susceptible host. The above said three
points are the derivation of Robert Koch.
Host – parasite
1. Pre-Penetration Stage:
During pre-penetration stage the pathogen (inoculum) on arrival on
the host surface interacts sharply with the surrounding environment and host
itself. The environment which is an aggregate of all external conditions
including temperature, moisture (relative humidity), light and the competing
microorganisms; affects the life and form of the pathogen of the inoculum.
The optimum environmental condition for ideal growth of a pathogen again
varies on the nature of the pathogen and the host surface. For example, the
development and abstraction of conidia are favored by high air temperature
and humidity in downy mildew.
Whereas in powdery mildew both the number of spores and their
germination are greater in bright sunlight. In cereal rusts uredospore’s
germinate at low temperature, but the infection process is delayed at this
temperature. Again soil pH plays a very vital role in the growth of bacterial
plant pathogen in the rhizosphere (area of soil immediately surrounding the
roots). Whether a pathogen will survive and grow on the host surface also
depends on its behavior with the exudates of, the host surface and the
microbial population present on it. The exudates of the host surface may
encourage or inhibit the growth of the pathogen. The root exudates mainly
sugars and amino acids are nutrients for the growth of fungi and bacteria.

239
 But root exudates like hydrocyanic acid, various organic acids and
antibiotics are antifungal and antibacterial. For example, spores of Rhizopus
arrhizus germinate only in presence of proline (amino acid) present in the
rhizosphere region; whereas exudates of root of onion varieties inhibit spore
germination of Colletotrichum circinaus. Leaves also exude substances
which may go in favor or against the growth of the pathogen. The glands of
leaf hairs of gram contain malic acid which is antifungal and arrests the
growth of Uromyces ciceris arietini.
Protocatechuic acid, an exudate of onion skin is also antifungal. The
pathogen has to neutralize these exudates or has to be resistant to them for
survival. Besides these, the rhizosphere region contains microbial population
which is antagonistic to the growth of the pathogen. As such, the pathogen
has to overcome the above barriers during pre-penetration stage before it
can survive for host penetration. Once a favorable relationship is established
with the host surface, multiplication or growth of the pathogen begins. Rapid
proliferation of cells of bacterial pathogen results in a relatively short time.
The bacterial cells so formed being delicate structures may be easily killed
by unfavorable conditions.
Hence they survive under layers of slime. Again development of
fungal phytopathogen usually includes spore germination by germ tube
which grows producing infection hypha as a result of hyphal tip growth or
may give rise to an aspersorium which anchors the fungus to the host
surface. Penetration peg is produced from the aspersorium with which the
pathogen causes host penetration. But multiplication of viruses takes place
only in the living host cells.
2. Penetration Stage:
The success of host penetration leading to disease development is a
very complicated process which is a combined effect of various factors like:
(i) The nature and behavior of the pathogen including its multiplication
capacity,
(ii) Favorable physical conditions, and
(iii) Host susceptibility.

240
 Of all these factors, the factor nature and behavior of the pathogen is
the most important one which controls the overall disease development. The
nature and behavior of pathogen encompass the inoculum potential of the
pathogen. The inoculum potential is again a measure of the biological
energy available for the colonization of a host.
It is a function of:
(i) Inoculums density which refers to the number of viable propagules per
unit area of leaf or stem or per unit volume of soil;
(ii) The nutrients available to the infectious units that allow them to germinate
or grow;
(iii) The environment (temperature range of 15 to 25°C, moisture content 70
per cent, and relative humidity 90 to 95 per cent);
(iv) The virulence (aggressiveness) or genetic capacity of the pathogen to
cause disease; and
(v) The susceptibility of the host.
Besides these, the physiological state of the host may have an effect
on the ability of the pathogen to attack it or on the extent to which a
pathogen may harm it. The concept that encompasses this phenomenon is
termed predisposition.
2. Host penetration takes place:
(i) Through natural openings,
(ii) Through wounds,
(iii) By direct penetration of surface cells causing tissue disintegration, and
(iv) Through specific parts or organs.
Both bacteria and viruses enter the host tissue mainly through
wounds. Whereas, the fungal pathogens gain entrance in the host through
natural openings, wounds and by direct penetration through cuticle and outer
wall of the surface cells, or root hairs, or through specific parts or organs of
the host. The direct penetration process is more complicated than the entry
through natural openings and wounds. It is performed by breaking the host
structural barriers through wide range of chemical actions.
(i) Entry through natural openings:

241
 Both bacteria and fungi gain entrance into the host through natural
openings such as: stomata, lenticels, hydathodes, nectaries, leaf scars,
stigma, etc. This is a process in which the pathogens have an easy access
to the host, except in cases where sub-stomatal hairs may cause resistance
against the host entry.
(ii) Entry through wounds:
Wounds caused due to natural calamities (storm, fire, etc.); during
field operations; by insects, by accidental breaking of parts or otherwise;
offer easy passages of pathogens in the host. But so far as viruses are
concerned, the host entry is only through wounds.
(iii) Entry by direct penetration of surface cells:
The entry of pathogen by direct penetration of the outer wall of the
host surface cells is rather a difficult process for which the pathogen usually
requires high moisture or free water supply. It is even more difficult in leaves
with waxy covering on their surface which allows water to rim off freely.
Fungal pathogens penetrate into host either by boring through the
outer wall of the surface cell or penetration is effected by pressure and
sometimes due to chemical softening or solubilizing of the barrier caused by
the solvent action of enzymes secreted by the infecting organ. After the
hypha made contact with a suitable host surface, some growth in close
contact with it takes place. This is followed by the development of an
aspersorium or increase in diameter of hypha serving as adherent area from
which develops penetration tube. The penetration tube penetrates through
the cuticle at a point softened by enzymatic action and followed by
mechanical pressure. In some cases germ lube produced by spore
germination passes down between the racial wall of the adjoining cells
without actually entering the cells.
Other fungi send only haustona in the epidermal cell through the
outer cutinized wall In certain others, the penetration hypha penetrating the
cuticle induces the formation of a local interna swelling of the underlying
cellulose wall, so that a small papilla projects from the underside of the wall
into the cell. This papilla is penetrated by the penetration hypha which enters
the host cell and ultimately develops into a haustorium.

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 Bacteria are mostly weak parasites which cannot employ force to
effect penetration. Their penetration is effected by chemical action. The plant
parasitic nematodes pierce the host surface with spears or stylet.
3. Post-Penetration Stage:
Usually with the success of the penetration process, post-penetration
is successful. But the entry of the pathogen in the host tissue may not
always ensure immediate infection leading to disease development. The
process may be delayed or there may be failure for various reasons.
The delay may be in cases where the pathogen has incubation
period and infection is established only after the expiry of the incubation
period. The success of post-penetration process depends largely upon
competition of pathogen for nutrition, and production of enzymes and toxic
substances and their effects on host metabolic activities. Again due to toxic
effect of host cytoplasm, the pathogen may fail to establish biological
relationship with the host. Host-pathogen interaction may also result
hypersensitivity of the host tissue, whereby rapid death of the affected cells
prevents the further spreading of the pathogen due to shortage of nutrition.
But in most of the plant diseases, host infection is followed by invasion, a
condition when a pathogen grows rapidly in the host tissue. For example,
bacteria invade host tissues intracellular and destroy them. Whereas, fugal
mycelia invade inter- or intracellular but may or may not cause destruction
immediately after invasion. Viruses always invade host tissues intracellular.
They multiply in the living host cells by directing them to manufacture viral
nucleic acid and viral protein, their movement from cell to cell is through
plasmodesmata. Again fungal hyphae and spores, and bacterial cells may
move through vascular tissues once they gain entrance in them. Successful
host invasion of the pathogen is invariably associated with disease
syndromes of various types of varying degrees. After penetrating the host
cell walls, the pathogen comes in contact with the host cytoplasm from which
it gets its required nutrition. In response to the activities of the pathogen, the
host metabolic processes (osmoregulation, respiration, photosynthesis, etc.)
get upset.

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LET US SUM UP
Plants represent a rich source of nutrients for many organisms
including bacteria, fungi, protists, insects, and vertebrates. Although lacking
an immune system comparable to animals, plants have developed a
stunning array of structural, chemical, and protein-based defenses designed
to detect invading organisms and stop them before they are able to cause
extensive damage.

CHECK YOUR PROGRESS

1. --------------------Defined inoculums
2. Koch.s Pastulate defined by ------

GLOSSARY
1. Inoculum density – No of viable probagules

SUGGESTED READINGS
1. Plant Pathology by George N. Agrios

ANSWERS TO CHECK YOUR PROGRESS

1. Garrett
2. Robert Koch

MODEL QUESTIONS

1. Explain Koch’s Pastulate

244
UNIT – 21
STRUCTURAL AND BIOCHEMICAL
DEFENSE MECHANISMS IN PLANTS
STRUCTURE

Overview

Learning Objectives
21.1 Structural Defense

21.2 Biochemical Defense

Let us Sum up
Check your Progress
Glossary
Suggested Readings
Answers to check your progress
Model questions

OVERVIEW
Plants represent a rich source of nutrients for many organisms
including fungi, bacteria, virus, nematodes, insects, and vertebrates. Plants
have developed a stunning array of structural, chemical, and protein- based
defenses designed to detect invading organisms and stop them before they
are able to cause extensive damage.

LEARNING OBJECTIVES

To Understand the plant defense mechanism

21. I STRUCTURAL DEFENSE:

In plants some structures are already present to defend the attack
while in others, the structures to defend the host develops after the infection.

245
In this way, structural defense can be characterized as (A) Preexisting
defense structures and (B) Defense structures developed after the attack of
the pathogen.
(A) Preexisting Defense Structures:
(i) Cuticular Wax:
Wax-mixtures of long chain aliphatic compounds get deposited on
the cuticular surface of some plants. Deposition of wax on the cuticular
surface is thought to play a defensive role by forming a hydrophobic surface
where water is repelled.
As a result, the pathogen does not get sufficient water to germinate
or multiply. In addition, a negative charge usually develops on the leaf
surface due to the presence of fatty acids – the main component of cuticle.
The negative charge prevents/reduces the chance of infection by many
pathogens.
(ii) Cuticle Thickness:
The thickness of cuticle is most important for those which try to enter the
host through the leaf surface. The cuticle thickness obstructs the path of
pathogen. In addition, a thick cuticle checks the exit of the pathogen from
inside the host, thus reducing the secondary infection.
(iii) Structure of Epidermal Cell Walls:
Tough and thick outer walls of epidermal cells may directly prevent
the entry of the pathogen completely or make the entry difficult. The
presence or absence of lignin and silicic acid in the cell walls may show
variation in resistance to penetration of the [Link] outer walls of
epidermal cells of rice plants are lignified and are seldom penetrated by blast
disease of rice pathogen. In resistant varieties of potato tubers (resistant to
Pythium debaryanum) the epidermal cells contain higher fibre content than
the susceptible ones.
(iv) Structure of Natural openings:
Structure of natural openings like stomata lenticels etc. also decide
the fate of the entry of the pathogen. In Szincum variety of citrus, the
stomata are small and possess very narrow openings surrounded by broad
lipped raised structures which prevent entry of water drops containing citrus

246
canker bacterium. In the same way, the size and internal structures of
lenticels may play a defensive role against the pathogens. Varieties having
small lenticels in the apple fruits prevent the entry of the pathogen while
those having large openings easily allow the pathogen to enter. Nectaries
provide openings in the epidermis and may play a defensive role due to high
osmotic concentration of the nectar. In resistant varieties of apple, presence
of abundant hairs in the nectaries acts as a defense mechanism while
susceptible varieties are devoid of abundant hairs.
Internal Defense Structures:
There are many preexisting internal defense structures inside the
plant that prevent the entry of pathogen beyond these structures. In some
plants, cell walls of certain tissues become thick and tough due to
environmental conditions and this makes the advance of the pathogen quite
difficult.
In case of stems of cereal crops, vascular bundles or extended areas
of sclerenchyma cells checks the progress of rust pathogen. Leaf veins
effectively obstruct the spread of pathogen like the angular leaf spot
pathogen.
(B) Defense Structures Developed after the Attack of the Pathogen:
After the pathogen has successfully managed to overcome the preexisting
defense mechanisms of the host, it invades the cells and tissues of the host.
In order to check the further invasion by the pathogen, the host plants
develop some structures/mechanisms which may be defense reactions in
the cytoplasm, cell wall defense structures, defense structures developed by
the tissues and ultimately the death of the invaded cell i.e. necrosis. These
will be briefly discussed here.
(I) Defense Reactions in the Cytoplasm:
The cytoplasm of the invaded cell surrounds the hyphae of the pathogen and
the nucleus of the host cell gets stretched to break into two. In some host
cells, the cytoplasm and the nucleus of the infected cells enlarge. The
cytoplasm becomes granular and dense and develops granular particles.
These result in the disintegration of the pathogen mycelium and thus the

247
invasion stops. Such cytoplasmic defence mechanisms can be seen in weak
pathogens like Annillaria and some mycorrhizal fungi.
(II) Cell Wall Defense Structures:
Cell wall defense structures are of limited help to the host. These include
morphological changes in the cell wall of the host.
Three types of cell wall defense structures are generally observed:
(i) Cell walls thicken in response to the pathogen by producing a cellulose
material, thus preventing the entry of the pathogen
(ii) The outer layer of cell walls of the parenchyma cells in contact with
invading bacterial cells produce an amorphous fibrillar material that traps the
bacteria thus preventing them to multiply and
(iii) Callose papillae get deposited on the inner layers of the cell walls due to
invasion by fungal pathogens.
In raw cases, the hyphal tips of the infecting fungal pathogen penetrating the
cell wall and thereafter growing into the cell lumen get enveloped by callose
material that, later become infused with phenolics forming a sheath around
the hyphae.
(III) Defense Structures Developed by the Tissues:
The following four developments take place in the tissues after
penetration:
(a) Gum Deposition:
Plants produce a variety of gummy substances around lesions or
spots as a result of infection. These gummy substances inhibit the progress
of the pathogen. The gummy substances are commonly produced in stone
fruits.
(b) Abcission Layers:
Abscission layers are usually formed to separate the ripe fruits and
old leaves from the plant. But in some stone fruit trees, these layers develop
in their young leaves in response to infection by several fungi, bacteria or
viruses. An abscission layer is a gap formed between two circular layers of
cells surrounding the point of infection. This gap is created by the dissolution
of one or two layers of the middle lamella, one or two layers of cells
surrounding the infected loci resulting in the infected locus becoming

248
unsupported, shrivels, dies and falls down along with the pathogen.
Abscission layer formation protects the healthy leaf tissue from the attack of
the pathogen.
Di

Fig. 21.1 Different defense structure after infection A” Gum, B: Abscission

layer, C: Tyloses, D: Sheath, E, F: Cork layer
(C) Tyloses:
Tyloses are out growths of protoplasts of adjacent live parenchyma
cells protruding into xylem vessels through pits under stress or in response
to attack by the vascular pathogens. Their development blocks the Xylem
vessels, obstructing the flow of water and resulting in the development of wilt
symptoms. However, tyloses are formed in some resistant plants ahead of
infection and the prevent the plant from being attacked.
(D) Formation of Layers:
Some pathogens like certain bacteria, some fungi and even some
viruses and nematodes stimulate the host to form multilayered cork cells in

249
response to infection, these develop as a result of stimulation of host cells by
substances secreted by thus, pathogen. These layers inhibit the further
invasion by the pathogen and also block the flow of toxic substances
secreted by the pathogen. Cork layers also stop the flow of nutrients of the
host thus also depriving the pathogen of the nutrients. Examples of cork
layer formation as a result of infection are: soft not of potato caused by
Rhizopus sp., potato tuber disease caused by Rhizoctonia sp., Scab of
potato caused by Streptomyces scabies and necrotic lesions on tobacco
caused by tobacco mosaic virus.
IV. Necrosis or Hypersensitive Type of Defense:
Necrosis or hypersensitive type of defense is another defense
mechanism adopted by some pathogens like Synchytrium endobioticum
causing wart disease of potato, Phytophthora infestans causing late blight
disease of potato and Pyricularia oryzae causing blast of rice etc. In such
diseases, the host nucleus moves toward the pathogen when the latter
comes in contact with the protoplasm of the host. The nucleus soon
disintegrates into brown granules which first accumulate around the
pathogen, later dispersing throughout the host cytoplasm. Soon the cell
membrane swells and finally the cell bursts and dies. These cause the
pathogen nucleus to disintegrate into a homogenous mass and its cytoplasm
dense. As a result, the pathogen fails to grow beyond the necrotic or dead
cells and the further growth of the pathogen is stopped.
21.2 Biochemical Defense:
Although structural defense mechanisms do prevent the attack of the
pathogen, the defense mechanism also includes the chemical substances
produced in the plant cells before or after the infection. It has now been
established that biochemical defense mechanisms play more important role
than the structural defense mechanisms. This has been supplemented by
the fact that many pathogens entering non host plants naturally or artificially
inoculated fail to cause infections in absence of any structural barriers. This
does suggest that chemical defense mechanisms rather than structural
mechanisms are responsible for resistance in plants against certain
pathogens.

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(A) Preexisting Biochemical Defense:
(i) Inhibitors Released in the Prepenetration Stage:
Plant generally exudes organic substance through above ground parts
(phyllosphere) and roots (rhizosphere). Some of the compounds released by
some plants are known to have an inhibitory effect on certain pathogens
during the prepenetration stage. For example fungistatic chemicals released
by tomato and sugar beet prevent the germination of Botrytis and
Cercospora. Presence of phenolics like protocatechuic acid and catechol in
scales of red onion variety inhibit the germination of conidia of Colletotrichum
circinans on the surface of red onion. Inhibitors present in high
concentrations in the plant cells also play an important role in defense of
plants. Presence of several phenolics, tannins and some fatty acid like
compounds such as dienes in cells of young fruits, leaves or seeds afford
them resistance to Botrytis. The tubers of resistance vars of potato against
potato scab disease contain higher concentrations of chlorogenic acid
around the lenticels and tubers than the susceptible vars. Several other
compounds like saponin tomatin in tomato and avinacin in oats have
antifungal activity. Some enzymes like glucanases and chitinases present in
cells of some plants may break down the cell wall components of pathogens.
(ii) Lack of nutrients essential for the pathogen is another preexisting
biochemical defense mechanism. Plant varieties or species which do not
produce any of the chemicals essential for the growth of pathogen may act
as resistant variety. For example, a substance present in seedling varieties
susceptible to Rhizoctonia initiates hyphae cushion formation from which the
fungus sends penetration hyphae inside the host plants. When this
substance is not present, hyphal cushions are not formed and the infection
does not occur.
(iii) Absence of Common Antigen in Host plant:
It is now clear that the presence of a common protein (antigen) in both the
pathogen and host determines diseases occurrence in the host. But if the
antigen is present in the host and absent in the host or vice-versa, it makes
the host resistant to the pathogen. For example, varieties of linseed which
have an antigen common to their pathogen are susceptible to the disease

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rust of linseed caused by Melampsora lini. In contrast, the absence of
antigen in linseed varieties but occurring in the pathogen are resistant to the
pathogen. Another example is leaf spot disease of cotton caused by
Xanthomonas campestris pv. malvacearum.
(B) Post-Infection-Biochemical Defense Mechanism:
In order to sight infections caused by pathogens or injuries caused by
any other means, the plant cells and tissues produce by synthesis many
substances (chemicals) which inhibit the growth of causal organism. These
substances are generally produced around the site of infection or injury with
the main aim at overcoming the problem.
Some such important chemicals are described below:
(i) Phenolic Compounds:
These are the most common compounds produced by plants in
response to injury or infection. The synthesis of phenolic compounds takes
place either through “acetic acid pathway” or “Shikimic acid pathway”. Some
common phenolic compounds toxic to pathogens are chlorgenic acid, caffeic
acid and ferulic acid. These phenolic compounds are produced at a much
faster rate in resistant varieties than in susceptible varieties. Probably that
the combined effect of all phenolics present is responsible for inhibiting the
growth of the infection.
(ii) Phytoalexins:
Phytoalexins are toxic antimicrobial substances synthesized ‘de
novo’ in the plants in response to injury, infectious agents or their products
and physiological stimuli. The term phytoalexin was first used by the two
phytopathologists Muller and Borger (1940) for fungi static compounds
produced by plants in response to mechanical or chemical injury or infection.
All phytoalexins are lipophilic compounds and were first detected after a
study of late blight of potato caused by Phytophthora infestans. Phytoalexins
are believed to be synthesized in living cells but surprisingly necrosis follows
very quickly.
According to Bill (1981), peak concentration of phytoalexins almost
always coincides with necrosis. Although the exact mechanism of production
of phytoalexin has not been properly understood, it is considered that a

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metabolite of the host plant interacts with specific receptor on the pathogen’s
membrane resulting in the secretion of “phytoalexin elicitor” which enters the
host plant cells and stimulates the phytoalexin synthesis. Phytoalexins are
considered to stop the growth of pathogens by altering the plasma
membrane and inhibiting the oxidative phosphorylation. Phytoalexins have
been identified in a wide variety of species of plants such as Soyabean,
Potato, sweet potato, barley, carrot, cotton etc. are being investigated. Some
common phytoalexins are Ipomeamarone, Orchinol, Pistatin, Phaseolin,
Medicarpin, Rishitin, Isocoumarin, ‘Gossypol’ Cicerin, Glyceolin, Capisidiol
etc.
The following Table gives a list of phytoalexins, chemical nature
the host and the pathogens in response to which these are produced:

(iii) Substances Produced in Host to Resist Enzymes Produced by

Pathogen:
Some hosts produce chemicals which neutralise the enzymes produced by
pathogen, thus defending the host. Therefore these substances help plants
to defend themselves from the attack of the pathogen.
In bean plants, infection with Rhizoctonia solani causes necrosis. In
resistant bean varieties, the entry of pathogen causes the separation of
methyl group from methylated pectic substances and forms polyvalent

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cations of pectic salts which contain calcium. The calcium ions accumulate in
infected as well as neighbouring healthy tissues and because of the calcium
accumulation, the pathogen fails to disintegrate middle lamella by its
polygalacturonase enzymes. These are known to dissolve the middle lamella
of healthy tissue in susceptible varieties.
(iv) Detoxification of Pathogen Toxins and Enzymes:
In some cases, the plants produce chemicals which deactivate the
toxins produced by the pathogens. For example, Pyricularia oryzae which
causes blast disease of rice produces Picolinic acid and pyricularin as toxins.
Although resistant varieties convert these toxins into N-methyl
picolininic acid pyrecularin into other compounds, the susceptible varieties
do get affected by these toxins. Similarly in case of cotton and tomato wilts,
the toxin fusaric acid produced by the pathogen gets converted into non-
toxic N-methyl-fusaric acid amide in resistant varieties. As in case of
detoxification of toxins, the toxic enzymes produced by the pathogen is
deactivated by phenolic compounds or their oxidation products. Some
varieties of cider apple are resistant to brown not disease caused by
Sclereotinia fructigena. It may be because of the resistant varieties
producing pheolic oxidation products which inactivate the pectinolytic
enzymes produced by the pathogen.
(v) Biochemical Alterations:
It has been observed that infection of the host by the pathogen brings
about biochemical changes in the host which may prove toxic to the
pathogenic microorganisms and cause resistance to the pathogen.
Production of certain new enzymes and other compounds are synthesized
and accumulated in higher concentration. This may also add to the
resistance of the plant by being toxic to pathogenic microorganisms.

LET US SUM UP

254
 Different types of chemical and structural defense mechanism
provide plant defenses response to the pathogens. Defense mechanism will
not work, when compatible reaction occurs between host and pathogen

CHECK YOUR PROGRESS

1. Cuticular surface provides ----------------------- surface.

2. -------------- toxic antimicrobial substances synthesized by

plants.

GLOSSARY

1. Necrosis - hypersensitive type of defense

SUGGESTED READINGS

1. Plant Pathology by P.D. Sharma

ANSWERS TO CHECK YOUR PROGRESS

1. Hydrophobic

2. Phytoalexins

MODEL QUESTIONS
1. Explain Biochemical Defense Mechanism

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UNIT 22
DEFENSE RESISTANCE AND
INCORPORATION OF RESISTANT
GENES

STRUCTURE

Overview

Learning Objectives
22.1 Genetic Virulence

22.2 Gene for gene relationship

Let us Sum up
Check your Progress
Glossary
Suggested Readings
Answers to check your progress
Model questions

OVERVIEW
Plants represent a rich source of nutrients for many organisms
including bacteria, fungi, protists, insects, and vertebrates. Although lacking
an immune system comparable to animals, plants have developed a
stunning array of structural, chemical, and protein-based defenses designed
to detect invading organisms and stop them before they are able to cause
extensive damage.

LEARNING OBJECTIVES
To understand the defense of plants

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22.1 GENITIC VIRULANE
A molecular cloning and characterization of ‘R’ (Resistance) genes
have been analysed after 1992. In the mechanism of disease resistance,
there are specific ‘R’ genes presence in the plant host and corresponding
virulence genes in the pathogen. Although nature containing vast array of
potential pathogen, only few plants exhibit resistance. Plant exhibit
resistance to pathogens in number of ways. They are hypersensitive
response (HR), localized induce cell death in the host plant at the site of
infection. Hypersensitive responses are prominently checking the growth of
pathogen. Genetic basis of HR-mediated resistance has been recorded by
H. H. Flor. He proved that disease resistance of flax to the fungal pathogen
Melampsora lini is due to the interaction between pair of genes in the host
and the pathogen. His work provides theoretical basis for molecular cloning
of pathogen avirulence (avr) genes and their corresponding plant R genes.
In the gene for gene interaction during plant defence, avirulence (avr) genes
of pathogen are expressed to produce elicitors.
These elicitors are signal molecules recognised by host plant and
leads to the cascade of hypersensitive reaction. The R genes of host plants
are presumed to encode receptors for these elicitors. The elicitor signal
molecule triggers the expression of a cascade of host genes that led to
hypersensitive reaction and check pathogen growth.

257
 Fig. 22.1 Diagramatic representation of gene for gene interaction.

Gene for gene interaction pathway shows involvement of signal

transduction. Some of the hypersensitive reaction (HR) which is common to
different pathogen include rapid oxidative burst, ion flux by K+H+ exchange,
cellular decomposition, cross linkage and strengthening of cell wall. In
addition, induction of pathogenesis related (PR) proteins takes place in
response to pathogen attack. Several crop plants such as tomato, bean,
potato and barley have been used to study crop responses to pathogen
attack. Based on the receptor signal model it was postulated that pathogen
avr gene specify elicitor signal molecule induce disease resistance in host
plant that contain ‘R’ genes

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 Fig. 22.2 Representation of receptor- Ligand model
In one of the case study, the interaction between leaf mold pathogen
Cladosporium fulvum and tomato plant have been well established. The two
avirulence genes, avr9 and avr4, belongs to C. fulvum encode precursor of
elicitor peptide that specifically induce HR response in plant that encodes
corresponding ‘R’ genes Cf-9 and Cf-4 respectively. In another confirmed
case, the avirulence gene avr D of Pseudomonas syringae encodes
enzymes involved in the synthesis of syringolids that elicit HR in soyabean
carrying R gene Rpg4. Similarly, TMV coat protein functions as intracellular
elicitor that induce HR in Nicotiana sylvestris carry corresponding gene
known as N, these studies reveals that having a avirulence has definite
advantage to pathogen and that can enhance virulence on host (susceptible)
that do not carry ‘R’ genes.
22.2 GENE FOR GENE RELATIONSHIP:
Several R genes have been isolated and characterized by positional
cloning and transposon tagging. The classic gene for gene relationship was
characterized in tomato pto gene. The pto gene confers resistance to P.
syringae (pst) carrying avirulence gene avr pto. Expressed product of pto
gene confirmed that it encodes a serine-threonine protein kinase. This
enzyme probably involved in signal transduction. The fine locus of PTO
consists of cluster of five to seven genes, all homologous to PTO. Some of

259
these genes like FEN, PRF and others conferred resistance and sensitivity
to organophosphorus insecticide fenthion.
In another case study, four ‘R’ genes have been cloned to confirm
the concept of gene-for- gene relation, for example Arabidopsis carrying over
RPS2 genes, which confer resistance to pathogen P. syringae carries avr
RPS2. The lack of RPS2 ‘R’ gene in mutant Arabidopsis result in complete
absense of HR during pathogen (P. syringae) attack carries RPt2.
The RPS2 gene was then cloned by map-based strategy to initiate HR
process. Transposon tagging helps in the identification of several resistance
gene like flax L6, tobacco N and tomato cf-9 gene. Translation product of
RPS2, N, cf-9 and L6 R genes revealed that their proteins contain leucine,
rich repeat (LRR). These four genes are different from both maize HM, and
the tomato pto resistance (R) genes. RPS2, N, and L6 except cf-9 sharing
some homology and contain conserved nucleotide binding site (NBS). The
sequence of rice Xa21 R gene, which confers resistance to Xanthomonas
oryzae race 6, predicted that their protein also conferring both leucine rich
repeat motif and serine-threonine-kinase like domain suggested a role in cell
surface recognition of pathogen ligand and subsequent activation of an
intracellular defence response characterization of Xa21 gene lead to
engineered resistance in rice.
Further studies on ‘R’ proteins revealed that Cf 9 protein appears to
consist of primarly of extra cytoplasmic LRR and also suggested that Cf-9 is
a receptor for extracellular ligand provided by the avr 9 elicitor peptide of
pathogen. Another R protein RPS2 contains leucine zipper at NH2-terminus,
probably involved in protein dimerization. The isolation of over 30 plant
disease resistance genes revealed that most genes encode putatively
cytoplasmic proteins with a nucleotide binding site (NBS) and a leucine-rich
repeat (LRR). However, the recent isolation of the Arabidopsis RRSI-R gene
has NBS-LRR subtype that contains αC-terminal extension with a putative
nuclear localization signal and a DNA binding domain.
Deviation in gene-for-gene resistance has also been established for
example, HMI gene in maize, which control resistance to Cochliobolus
carbonum, HMI encodes NADPH dependent HC toxin reductase, which

260
inactivate HC toxin, pathogenecity factors, produced by fungus C. carbonum.
The genetic interaction between maize and pathogen fungus different from
gene for gene interaction because toxin-deficient C. carbonum fail to impose
disease in maize that do not carry HMI.

LET US SUM UP
The mechanisms discussed in this article represent a broad overview
of plant defense responses. In this Unit we discussed about the resistance of
disease at molecular level.

CHECK YOUR PROGRESS

1. Example for gene interaction is -------------
2. Gene for gene is characterized in -------

GLOSSARY
1. Elicitors - elicitors are signal molecules recognised by host plant

SUGGESTED READINGS
1. Introduction to Principles of Plant Pathology by R. S. Singh

ANSWERS TO CHECK YOUR PROGRESS

1. Melampsora lini
2. tomato pto gene

MODEL QUESTIONS
1. Write about Gene for gene concept.

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UNIT 23
GENE TRANSFER SYSTEM
STRUCTURE

Overview

Learning Objectives
23.1 Direct Gene Transfer System
23.2 Natural mode of Gene Transfer
Let us Sum up
Check your Progress
Glossary
Suggested Readings
Answers to check your progress
Model questions

OVERVIEW
Gene transfer carried in two methods into plants, one is natural mode
another one is artificial mode. Artificial mode of gene transfer carried out in
plants by many techniques such as Electroporation, Micro injection,
Chemical mediated gene delivery and Particle bombardment. In natural
mode, transformation and transduction are the two methods used. This unit
discusses the above said methods.

LEARNING OBJECTIVES

To understand the gene transfer techniques in a detailed manner.

23.1 DIRECT GENE TRANSFER SYSTEM:

It may also be called as DNA mediated Gene Transfer (DMGT) or
vector less gene transfer system. This system does not involve the use of
any biological agent like Agrobacterium. The DNA is introduced directly into
the plant cell by using certain chemical or physical treatments.

262
Main methods for direct gene transfer are given below:
(i) Electroporation:
Short electric pulses of high voltage are given to the target cells
which results in the formation of temporary small pores in the cell
membrane, these pores allow the intake of DNA into the protoplast of cells.
(Fig.23 1).

Fig. 23.1 Electroporation

(ii) Chemical Methods:
Some chemical helpers are also employed for stimulating direct gene
delivery. PEG (Poly Ethylene Glycol) is one such chemical stimulant. PEG
precipitates the DNA on outer surface of plasma membrane of target cells

263
and this precipitate is then taken in by endocytosis thus resulting in the entry
of DNA into the cell. Calcium phosphate is another example of chemical
helper for direct gene transfer.
(iii) Micro-injection:
It is the introduction of new DNA into the target cell by injecting it
directly into the nucleus or the protoplast. The target cells are immobilized on
a solid surface and the micromanipulator is used for microinjecting the DNA
into the cell (Fig. 23.2).

Fig.23.2 Microinjection
(iv) Particle-Gun Method:
It is also called micro-projectiles or biolistic method or ballistic
method. It involves the bombardment with high velocity micro projectiles onto
the target cells. The micro projectiles used are 1-3 mm diameter particles of
gold or tungsten coated with the DNA to be transferred (Fig. 23.3).

264
 Fig. 23.3 Particle bombardment

23.2 NATURAL MODE OF GENE TRANSFER

Agrobacterium-Mediated Gene Transfer:
Agrobacterium tumefaciens is a soil-borne, Gram-negative
bacterium. It is rod shaped and motile, and belongs to the bacterial family of
Rhizobiaceae. A. tumefaciens is a phytopathogen, and is treated as the
nature’s most effective plant genetic engineer. Some workers consider this
bacterium as the natural expert of inter-kingdom gene transfer. In fact, the
major credit for the development of plant transformation techniques goes to
the natural unique capability of A. tumefaciens. Thus, this bacterium is the
most beloved by plant biotechnologists.
Crown Gall Disease and Ti Plasmid:

265
 Almost 100 years ago (1907), Smith and Townsend postulated that a
bacterium was the causative agent of crown gall tumors, although its
importance was recognized much later. As A. tumefaciens infects wounded
or damaged plant tissues, in induces the formation of a plant tumor called
crown gall (Fig. 23.4). The entry of the bacterium into the plant tissues is
facilitated by the release of certain phenolic compounds (acetosyringone,
hydroxyacetosyringone) by the wounded sites.

Fig. 23.4 Formation of Crown Gall

Crown gall formation occurs when the bacterium releases its Ti

plasmid (tumor- inducing plasmid) into the plant cell cytoplasm. A fragment
(segment) of Ti plasmid, referred to as T-DNA, is actually transferred from
the bacterium into the host where it gets integrated into the plant cell
chromosome (i.e. host genome). Thus, crown gall disease is a naturally
evolved genetic engineering process.
Organization of Ti plasmid:
The Ti plasmids (approximate size 200 kb each) exist as independent
replicating circular DNA molecules within the Agrobacterium cells. The T-
DNA (transferred DNA) is variable in length in the range of 12 to 24 kb,
which depends on the bacterial strain from which Ti plasmids come.
Nopaline strains of Ti plasmid have one T-DNA with length of 20 kb while
octopine strains have two T-DNA regions referred to as TL and TR that are

266
respectively 14 kb and 7 kb in length. A diagrammatic representation of a Ti
plasmid is depicted in Fig.23,5 The Ti plasmid has three important regions.

Fig. 23.5 Ti Plasmid

T-DNA region:

This region has the genes for the biosynthesis of auxin (aux),
cytokinin (cyt) and opine (ocs), and is flanked by left and right borders.
These three genes-aux, cyto and ocs are referred to as oncogenes, as they
are the determinants of the tumor phenotype. T-DNA borders — A set of 24
kb sequences present on either side (right and left) of T-DNA are also
transferred to the plant cells. It is now clearly established that the right
border is more critical for T-DNA transfer and tumori-genesis.
Virulence region:

The genes responsible for the transfer of T-DNA into the host plant
are located outside T-DNA and the region is referred to as vir or virulence
region. Vir region codes for proteins involved in T-DNA transfer. At least nine
vir-gene operons have been identified. These include vir A, vir G, vir B1, vir
C1, vir D1, D2 and D4, and vir E1, and E2.
Opine catabolism region:
This region codes for proteins involved in the uptake and
metabolisms of opines. Besides the above three, there is ori region that is

267
responsible for the origin of DNA replication which permits the Ti plasmid to
be stably maintained in A. tumefaciens.
T-DNA transfer and integration:
The process of T-DNA transfer and it integration into the host plant
genome is depicted in Fig. 23.5 and is briefly described.
1. Signal induction to Agrobacterium:
The wounded plant cells release certain chemicals- phenolic
compounds and sugars which are recognized as signals by Agrobacterium.
The signals induced result in a sequence of biochemical events in
Agrobacterium that ultimately helps in the transfer of T-DNA of T-plasmid.
2. Attachment of Agrobacterium to plant cells:
The Agrobacterium attaches to plant cells through polysaccharides,
particularly cellulose fibres produced by the bacterium. Several
chromosomal virulence (chv) genes responsible for the attachment of
bacterial cells to plant cells have been identified.
3. Production of virulence proteins:
As the signal induction occurs in the Agrobacterium cells attached to
plant cells, a series of events take place that result in the production of
virulence proteins. To start with, signal induction by phenolics stimulates vir
A which in turn activates (by phosphorylation) vir C. This induces expression
of virulence genes of Ti plasmid to produce the corresponding virulence
proteins (D1, D2, E2, B etc.). Certain sugars (e.g. glucose, galactose, xylose)
that induce virulence genes have been identified.
4. Production of T-DNA strand:
The right and left borders of T-DNA are recognized by vir D1/vir
D2 proteins. These proteins are involved in the production single-stranded T-
DNA (ss DNA), its protection and export to plant cells. The ss T-DNA gets
attached to vir D2.
5. Transfer of T-DNA out of Agrobacterium:
The ss T-DNA — vir D2 complex in association with vir G is exported from
the bacterial cell. Vir B products form the transport apparatus.
6. Transfer of T-DNA into plant cells and integration:

268
 The T-DNA-vir D2 complex crosses the plant plasma membrane. In
the plant cells, T-DNA gets covered with vir E2. This covering protects the T-
DNA from degradation by nucleases; vir D2 and vir E2 interact with a variety
of plant proteins which influences T-DNA transport and integration.
The T-DNA-vir D2-vir E2 — plant protein complex enters the nucleus
through nuclear pore complex. Within the nucleus, the T-DNA gets
integrated into the plant chromosome through a process referred to
illegitimate recombination. This is different from the homologous
recombination, as it does not depend on the sequence similarity.
Hairy Root Disease of A. Rhizogenes — R1 Plasmids:
Agrobacterium rhizogenes can also infect plants. But this results in
hairy roots and not crown galls as is the case with A. tumefaciens. The
plasmids, of A. rhizogenes have been isolated and characterized. These
plasmids, referred to as Ri plasmids, (Ri stands for Root inducing) are of
different types. Some of the Ri plasmid strains possess genes that are
homologous to Ti plasmid e.g. auxin biosynthetic genes. Instead of virulence
genes, Ri plasmids contain a series of open reading frames on the T-DNA.
The products of these genes are involved in the metabolism of plant growth
regulators which gets sensitized to auxin and leads to root formation.
Vectors of A. rhizogenes:
As it is done with A tumefaciens, vectors can be constructed by using
A. rhizogenes. These vectors are alternate strategies for gene transfer.
However, employment of A. rhizogene-based vectors for plant
transformation is not common since more efficient systems of A. tumefaciens
have been developed.
Importance of hairy roots:
Hairy roots can be cultured in vitro, and thus are important in plant
biotechnology. Hairy root systems are useful for the production of secondary
metabolites, particularly pharmaceutical proteins.
Ti Plasmid-Derived Vector Systems:
The ability of Ti plasmid of Agrobacterium to genetically transform
plants has been described. It is possible to insert a desired DNA sequence

269
(gene) into the T-DNA region (of Ti plasmid), and then use A. tumefaciens to
deliver this gene(s) into the genome of plant cell.
Plant Transformation Technique Using Agrobacterium:
Agrobacterium-mediated technique is the most widely used for the
transformation of plants and generation of transgenic plants. The important
requirements for gene transfer in higher plants through Agrobacterium
mediation are listed.
i. The explants of the plant must produce phenolic compounds (e.g.
autosyringone) for activation of virulence genes.
ii. Transformed cells/tissues should be capable to regenerate into whole
plants.
In general, most of the Agrobacterium-mediated plant transformations have
the following basic protocol (Fig. 23.6).
1. Development of Agrobacterium carrying the co-integrate or binary vector
with the desired gene.
2. Identification of a suitable explant e.g. cells, protoplasts, tissues, calluses,
organs.
3. Co-culture of explants with Agrobacterium.
4. Killing of Agrobacterium with a suitable antibiotic without harming the plant
tissue.
5. Selection of transformed plant cells.
6. Regeneration of whole plants.
Advantages of Agrobacterium- mediated transformation:
i. This is a natural method of gene transfer.
ii. Agrobacterium can conveniently infect any explant (cells/tissues/organs).
iii. Even large fragments of DNA can be efficiently transferred.
iv. Stability of transferred DNA is reasonably good.
v. Transformed plants can be regenerated effectively.

270
Fig.23. 6 Transformation by Agrobacterium

271
Limitations of Agrobacterium- mediated transformation:
i. There is a limitation of host plants for Agrobacterium, since many crop
plants (monocotyledons e.g. cereals) are not infected by it. In recent years,
virulent strains of Agrobacterium that can infect a wide range of plants have
been developed.
ii. The cells that regenerate more efficiently are often difficult to transform,
e.g. embryonic cells lie in deep layers which are not easy targets for
Agrobacterium.
Virus-Mediated Gene Transfer (Plant Viruses as Vectors):
Plant viruses are considered as efficient gene transfer agents as they
can infect the intact plants and amplify the transferred genes through viral
genome replication. Viruses are natural vectors for genetic engineering.
They can introduce the desirable gene(s) into almost all the plant cells since
the viral infections are mostly systemic.
Plant viruses are non-integrative vectors:
The plant viruses do not integrate into the host genome in contrast to
the vectors based on T-DNA of A. tumefaciens which are integrative. The
viral genomes are suitably modified by introducing desired foreign genes.
These recombinant viruses are transferred, multiplied and expressed in plant
cells. They spread systemically within the host plant where the new genetic
material is expressed.

LET US SUM UP
Plant transformation was first described in tobacco in 1984. Since
that time, rapid developments in transformation technology have resulted in
the genetic modification of many plant species. Methods for introducing
diverse genes into plant cells include Agrobacteriumtumefaciens-mediated
transformation. Several gene transformation techniques utilize DNA uptake
into isolated protoplasts mediated by chemical procedures, electroporation,
or the use of high-velocity particles (particle bombardment). Direct DNA
uptake is useful for both stable transformation and transient gene

272
expression. However, the frequency of stable transformation is low, and it
takes a long time to regenerate whole transgenic plants.

CHECK YOUR PROGRESS

1. Electroporation is a ------------- gene transfer method.

2. For Transformation -------------------- is used.

GLOSSARY

1. Gall – undifferentiated mass of tumor cells

SUGGESTED READINGS

1. Plant Biotechnology: The Genetic Manipulation of Plants by

Slater

ANSWERS TO CHECK YOUR PROGRESS

1. Artificial

2. Agrobacterium

MODEL QUESTIONS
1. Write about Natural mode of Gene transfer.

273
274