MICROBIOLOGY FOR NURSING PROFFESIONALS
-Define Microbial Physiology:
Microbial physiology is an enormous discipline encompassing the study of thousands of
different microorganisms.
-Both living and non-living things are composed of molecules made from chemical elements such as
Carbon, Hydrogen, Oxygen and Nitrogen. The organization of these molecules into cells is one
feature that distinguishes living things from all other matter.
- The cell is the smallest unit of matter that can carry on all the processes of life. It is the basic
unit of all living things, and all organisms—from the tiniest bacterium to the largest whale—are
made up of one or more cells.
-In addition to having the same basic structure, all cells carry out similar life processes. These
include transport of materials, obtaining and using energy, waste disposal, replication
(reproduction), and responding to their environment.
-The idea that all living things are made of cells was put forward in about 1840 and in 1855 by three
German scientists, Theodor Schwann, a zoologist, Matthias Jakob Schleiden, a botanist, and
Rudolf Virchow, a medical doctor. They suggested that cells were the basic unit of all living
things. Rudolf further proposed that all cells arise only from other cells. The collective observations
of all three scientists led to the idea of cell theory.
Cell theory consists of four principles:
-The modern cell theory states that:
All organisms are made up of one or more cells.
All the life functions of an organism occur within cells (cells are the basic units of structure
and function in an organism; cells are units of metabolism).
All cells come from pre-existing cells (cells come only from the replication of existing cells).
Cells contain complete genetic material (DNA), hereditary substance.
Parts of a Cell:
There are many different types of cells, but all cells have a few features in common. These are: -
A cell or plasma membrane
-Cytoplasm
-Ribosomes for protein synthesis - DNA (genetic information).
The cell (plasma) membrane is the physical boundary between the inside of the cell
(intracellular) and its outside environment (extracellular). It acts almost like the “skin” of the
cell.
Cytoplasm is the general term for all of the material inside the cell. Cytoplasm is made up of
cytosol, a watery fluid that contains dissolved particles (e.g. proteins and lipids) and organelles.
Organelles are structures that carry out specific functions inside the cell. Ribosomes are the
organelles on which proteins are made. Ribosomes are found throughout the cytosol of the cell.
All cells also have DNA. DNA contains the genetic information needed for building structures
such as proteins and RNA molecules in the cell.
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Two Types of Cells:
-There are two cell types: prokaryotes and eukaryotes.
Prokaryotes (Greek: pro - before; karyon - nucleus) lack a well-defined nucleus and possess
relatively simple structure. These include the various bacteria, which are usually singlecelled
and smaller than eukaryotic cells.
Eukaryotes (Greek: eu - true; karyon - nucleus) possess a well-defined nucleus and are more
complex in their structure and function. Eukaryotic cells are usually found in multicellular
organisms (e.g. animals/humans and plants), but there are some single-celled eukaryotes [(e.g.
fungi e.g. yeast), protozoa and algae].
Prokaryotic Cells—General Features:
-Prokaryotes are organisms that do not have a cell nucleus or any organelles that are surrounded
by a membrane.
-Most of the metabolic functions carried out by a prokaryote take place in the plasma membrane.
-Prokaryotes have ribosomes, which are not surrounded by a membrane but do have a specialized
function, and could therefore be considered organelles.
Prokaryotic ribosomes are 70S (Svedberg units), which refers to their rate of sedimentation on
centrifugation and is a measure of their size, shape and density.
-Most prokaryotes are unicellular.
-Almost all prokaryotes have cell walls or cell envelopes located outside the cytoplasmic
membrane, which usually contain some peptidoglycan.
A cell wall gives structural support to the cell and acts as a barrier against outside forces.
-Some prokaryotes have an extra layer outside their cell wall called a capsule or slime coats, which
helps them stick to surfaces or to each other.
-Most prokaryotes have propelling flagella (singular: flagellum) that are less complex than those of
eukaryotic cells.
-Cell division in prokaryotes normally occurs by simple binary fission.
-Most prokaryotic cells contain a single circular chromosome composed of deoxyribonucleic acid
(DNA), which is located in a region of the cell referred to as the nucleoid. However, some
prokaryotes have linear chromosomes.
-Prokaryotic cells are very small; most are between 1-5 µm in diameter; however, some books refer
to prokaryotes as having diameters between 1-10 µm.
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Eukaryotic Cells—General features
-A eukaryote is an organism whose cells are organized into complex structures by internal
membranes and a cytoskeleton, as shown in the Figure below. The most characteristic
membrane-bound structure of eukaryotes is the nucleus. This feature gives them their name, which
comes from Greek and means “true nucleus”.
-The nucleus is the membrane-enclosed organelle that contains DNA. Eukaryotic DNA is organized
in one or more linear molecules, called chromosomes. Some eukaryotes are singlecelled, but many
are multicellular.
-In addition to having a plasma membrane, cytoplasm, a nucleus and ribosomes, eukaryotic cells
also contain membrane-bound organelles e.g. ribosomes, mitochondria, lysosomes, Golgi bodies
and an extensive endoplasmic reticulum.
-Eukaryotic ribosomes are 80S, somewhat larger than those of prokaryotes.
-Each organelle in a eukaryote has a distinct function. Because of their complex level of
organization, eukaryotic cells can carry out many more functions than prokaryotic cells. The
main differences between prokaryotic and eukaryotic cells are shown in Figures and Tables below.
-Eukaryotic cells may or may not have a cell wall. Plant cells generally have cell walls, while
animal cells do not. However, if cell walls are present, they are composed of materials other than
peptidoglycan, such as cellulose and related β-glucans, chitin or silica.
-Eukaryotic cells divide by a complex process of mitosis or meiosis.
Mitosis is a type of cell division for somatic cells and for the asexual reproduction of
unicellular eukaryotic cells while;
Meiosis (or reduction division) is the type of cell division for the production of gametes in
sexual reproduction. This process halves the number of chromosomes (i.e. reduces the number
of chromosomes) from diploid (2n) chromosome pairs to produce haploid (n) cells containing
a single set of chromosomes, facilitates genetic recombination and results in the formation of
gametes.
-Eukaryotic cells are about 10 times the size of a typical prokaryote; they range between 10 and
100 µm in diameter while prokaryotes range between 1 to 5 µm or 1 and 10 µm in diameter.
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Summary of the Differences between Prokaryotic and Eukaryotic Cell Structures
Prokaryotes Eukaryotes
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Typical organisms Bacteria Protista, fungi, plants, animals
Typical size Smaller cells (~ 1-10µm) Larger cells ~ 10-100µm
Type of nucleus Nuclear body; no nucleus Real nucleus with nuclear envelope
DNA
DNA is circular without histone
proteins
DNA is linear and associated with histone
proteins to form chromatins
Ribosomes
Ribosomes are small (70S),
suspended in cytoplasm
Ribosome are large (80S), suspended in
cytoplasm or attached to membranes
Cytoplasmic
structure
Very few structures; no
cytoskeleton
Highly structured by membranes; with
cytoskeleton
Cell movement
(motility)
Flagella / cilia made of proteins
known as flagellin
Flagella and cilia made of proteins known
as tubulin
Golgi bodies None Present
Endoplasmic
reticulum
None Present
Mitochondria None 1 - 100 (though RBC’s have none)
Respiratory
enzymes
Are bound to plasma membrane
Are located in mitochondria
Cell wall Usually based on peptidoglycan When present, based on cellulose or chitin
Chloroplasts None Present in algae and plants
Organization
Usually single cells (unicellular)
Single cells, colonies, higher multicellular
organisms with specialized cells
Cell division
Binary fission
(simple division)
Mitosis (normal cell replication)
Meiosis (gamete production)
Reproduction Always Asexual Can be asexual or sexual
Selected Prokaryotic Cell Structures:
1: Flagellum (plural: flagella)
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-A flagellum is an extremely thin hair like appendage or filaments used by bacteria for locomotion /
motility.
-It’s usually longer than a bacterial cell Types of flagellation
1. Monotrichous: bacteria with single flagellum on one side e.g. pseudomonas aeruginosa
2. Lophotrichous: bacteria with several/bunch/tuft of flagella on one side e.g. pseudomonas
flourescens
3. Amphitrichous: bacteria with flagella arising from two opposite sides (i.e. flagella
developing at both sides) e.g. spirillum serpens
4. Peritrichous: bacteria with flagella all around (i.e. flagella all over their surface) e.g.
Escherichia coli
2: (a) Fimbriae
-These are bacterial surface appendages that are involved in interactions with other cells but do not
provide locomotion.
-Fimbriae are small, bristle like fibres emerging from the surface of many bacterial cells and made
of fimbrine proteins.
-They stick to each other and to surfaces.
-They help in microbial colonization and attachments on surfaces like rocks and glass.
-Help pathogens to adhere to epithelial cells e.g. colonization of intestines by Escherichia coli and
genitourinary tract by gonococcus e.g. Neisseria gonorrhoeae.
-Fimbriae acts as a disease causing agents after attachment
(b) Pilli also known as sex pilus
-Is an elongate, rigid, tubular structure made of special protein known as pilin
-Pili are used for reproduction (“mating process”) between cells known as conjugation, which
involves partial transfer of DNA from one cell to another (in Gram-negative bacteria).
-A pilus from the donor cell unites with a recipient cell thereby providing a cytoplasmic connection
for making the transfer.
-The rest of the functions are same as fimbriae.
3: Capsule (Glycocalyx)
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-A viscous jelly like substance outside bacterial cell Functions of capsules:
1. Protects bacteria by making them resistant to phagocytosis by white blood cells (protection
against immune system so they can cause disease).
2. Also protecting of the cell wall from attacks by various kinds of antibacterial agents, e.g.
antibiotics, bacteriophages, colicines, lysozyme and other lytic enzymes.
3. Acts as a food reserve during depletion of food in the environment.
4. Increases virulence of bacteria / makes them more pathogenic or virulent.
5. Plays a role in synthesis of hydrolytic enzymes for food digestion e.g. Ruminococcus (in
ruminant animals).
6. Also used for attachment.
7. Serves as a good antigen-for bacterial identification.
4: Cell wall
-The cell wall is the layer that lies just outside the plasma membrane. It is 10-25 nm thick, strong
and relatively rigid, though with some elasticity, and openly porous, being freely permeable to
solute molecules smaller than 10 kDa in mass and 1 nm in diameter.
-The cell wall is composed of peptidoglycan (also known as murein player), which is a
glycoprotein (i.e. protein / carbohydrate complex).
-Bacteria can be classified as either Gram-positive (+ve) or Gram-negative (-ve) based on the
difference in their cell wall structures, components or composition.
-Cell walls can be distinguished on the basis of Gram’s stain:
a) Gram-positive bacteria – possess thick cell walls made of peptidoglycan (carbohydrate
polymers cross-linked by proteins), with no outer membrane. Such bacteria retain a purple
color when stained with a dye known as crystal violet, and are thus known as Gram-
positive (named after the Danish bacteriologist, Hans Christian Joachim Gram who
developed this staining procedure).
b) Gram-negative bacteria - possess double cell walls, with a thin inner wall of peptidoglycan
and an outer wall of carbohydrates, proteins, and lipids. Such bacteria do not stain purple
with crystal violet and are known as Gram-negative.
Functions of the cell wall:
1. To impart shape and rigidity to the cell—maintaining the characteristic shape of the
bacterium.
2. Protects bacteria from external environment.
3. It supports the weak cytoplasmic membrane against the high internal osmotic pressure of
the protoplasm (ranges from 5 and 25 atmosphere)—i.e.—it provides structural support to
prevent a bacterium from bursting, rupturing or collapsing because of changes in osmotic
pressure (i.e. when the water pressure inside the cell is greater than that outside the cell).
4. It’s important in bacterial cell division and multiplication 5. It also acts as an antigen and
provides protection to bacteria.
6. Provides staining characteristics to the bacterium.
7. Site of action of antibody and colicin. [colicins are a type of bacteriocin i.e. peptide and
protein antibiotics released by bacteria to kill other bacteria of the same species, in order to
provide a competitive advantage for nutrient acquisition].
8. Contains components that are toxic to host.
9. Also functions in interactions (e.g. adhesion) with other bacteria and with mammalian cells.
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10. Provide specific protein and carbohydrate receptors for the attachment of some bacterial
viruses (bacteriophages or phages).
5: Cell membrane
-A very thin (5-10 mm) flexible (elastic) sheet molded around the cytoplasm.
-Composed of lipid together with proteins (lipoproteins) and small amounts of carbohydrates.
Functions of cell membrane:
1. The membrane provides a site for energy reactions (ATP generation), nutrient processing and
synthesis.
2. Semipermeable membrane—controlling the inflow and outflow of metabolites to and from the
cytoplasm. It serves as a barrier through which materials enter and exit the cell.
3. Housing enzymes—which are involved in outer membrane synthesis, cell wall synthesis, and in
the assembly and secretion of extra-cytoplasmic and extracellular substances such as enzymes
and toxins into the extracellular environment.
4. Housing many sensory and chemotaxis proteins that monitor chemical and physical changes in
the environment
5. Cell motility.
6. Mediation of chromosomal segregation during replication (assists DNA replication).
6: Cell cytoplasm
-A dense, gelatinous solution surrounded by cell membrane. Its major component is water (7080%)
which serves as a solvent for a mixture of nutrients such as sugars, amino acids, other organic
molecules and salts.
-The components of cytoplasm serve as building blocks for cell synthesis or as sources of energy.
-It forms basis that hold different components or organelles such as chromosome, ribosomes,
granules, etc.
7: Bacterial chromosome and plasmids
-The hereditary material in most bacteria exists in the form of single circular strands of DNA
(bacterial chromosome). A few have linear or multiple chromosomes.
-Bacteria lack true nucleus; their DNA is not enclosed by a nuclear membrane but is aggregated in a
central area of the cell called nucleoid.
-Many bacteria contain other extra-chromosomal pieces of DNA called plasmids. These strands
exist as separate double stranded circles of DNA.
-During bacterial reproduction, the plasmids are duplicated and passed on to offspring
-The plasmids (extra-chromosomal genetic materials) are not essential to bacterial growth and
metabolism but they confer protective traits such drug resistance, metal tolerance, production
of toxins and enzymes.
- NB: Plasmids are an important agent in modern genetic engineering techniques because they can be
manipulated and transferred from one bacterial cell to another.
8: Ribosomes (70S)
-A bacterial cell contains small ribosomes (70S) made of RNA and proteins. They occur in chains
and are dispersed throughout the cytoplasm.
-Many are also attached to the cell membrane.
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-A ribosome is a combination of special type of RNA known as ribosomal RNA or rRNA (60%) and
protein (40%).
-They serve as the sites of protein synthesis.
9: Inclusion bodies or Granules - are storage bodies (reserve deposits of nutrients).
-They contain condensed, energy-rich organic or inorganic substances e.g. proteins, carbohydrates
(like glycogen) and lipids within special single-layered membranes.
-Inclusion bodies found in some aquatic bacteria (e.g. blue green, purple and green photosynthetic
bacteria) are gas vesicles that provide buoyancy and flotation.
10: Endospores
-Endospores are the specialized resting cells or highly resistant dormant structures produced by
Gram-positive bacteria when the environment is not suitable for the bacteria to be reproductive or
when essential nutrients are not available any more.
-Endospores help bacteria to withstand hostile conditions and facilitate their survival.
-These structures are called endospores because the spores are formed inside a bacterial cell
membrane.
-Endospore are produced by bacterial genus Bacillus and Clostridium, e.g. Bacillus anthracis,
Bacillus cereus, Bacillus subtilis, Clostridium tetani and Clostridium difficile etc.
-The bacteria have two-phase life cycle—a vegetative cell and an endospore.
-The vegetable cell is the metabolically active and growing phase (i.e. reproductive stage).
-When exposed to adverse environmental conditions, bacteria form an endospore by a process
termed sporulation.
-Spores denote resting stage which helps bacteria to overcome adverse environmental conditions
that are unfavorable for vegetative growth of cell.
-Spores are not a reproductive form and not a storage granule.
-These spores are resistant to bactericidal agents and adverse physical/environmental conditions (or
processes) such as heating, freezing, desiccation, chemicals or radiations which cause death of
vegetative cells and thus may threaten the existence of the organism.
-Endospores also provide a selective advantage for survival and dissemination of the bacterial
species that produce them.
Uses of Spores in Biotechnology/Modern Biology:
1. Importance in food, industrial, and medical microbiology: Endospores are of great practical
importance in food, industrial, and medical microbiology because of their resistance and the fact
that several species of endospore forming bacteria are dangerous pathogens. Endospore often
survive boiling for an hour or more. Therefore, autoclaves must be used to sterilize many
materials.
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2. Sterilization control: For proper sterilization, spores of certain species of bacteria are
employed as indicator, e.g. Bacillus stearothermophilus which is destroyed at a
temperature of 121°C for 10 to 20 minutes (same temperature and time as used in
autoclaving). Prior to its use, these spores may be kept in autoclave. Proper sterilization is
indicated by the absence of the spores after autoclaving.
3. Research: Spore formation is well suited for research on the construction of complex
biological structures.
Selected Animal and Plant Cell Structure—The Eukaryotic Cell
1: Cell membrane (Plasma Membrane) - present in plant and animal cells
-This is a phospholipid bilayer (a bilipid membrane) that is made up of protein subunits
and carbohydrates, with a characteristic semi permeability factor (i.e. it is the semi-
permeable membrane that is present within the cell wall).
-It surrounds the cell cytoplasm, thus enclosing its content.
- Functions of the plant cell (plasma) membrane:
In plant cells, the cell membrane separates the cytoplasm from the cell wall.
It has a selective permeability hence it regulates the contents that move in and out of the
cell. It also protects the cell from external damage and provides support and stability
to the cell.
It has embedded proteins which are conjugated with lipids and carbohydrates, along the
membrane, used to transport cellular molecules.
2: Cytoplasm - present in plant and animal cells
-This is a gel-like matrix lying just below the cell membrane, housing most of the cell
organelles e.g. plastids, mitochondria, central vacuoles, endoplasmic reticulum, Golgi
bodies, storage granules and lysosomes etc.
-It’s made up of water, enzymes, salts, organelles, and various organic molecules.
-It’s not classified as one of the cell’s organelles because it doesn’t possess major roles except
being a physical medium for holding and housing most of the complex cell’s interior
organelles and being a medium for transporting and processing cell molecules for
maintaining cell life.
3: Nucleus - present in plant and animal cells
-The nucleus is the information center of a cell. It is a specialized complex membrane-
bound organelle which is only present in eukaryotic cells and whose primary function is to
store the cell’s genetic information.
-It is also responsible for coordinating the cell’s activities including cell metabolism, cell
growth, synthesis of proteins and lipids and generally the cell reproduction by cell division
mechanisms.
-
-
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-The nucleus contains the cells’ genetic information known as deoxyribonucleic acid (DNA)
on the chromosomes.
-Chromosomes are special thread-like strands of nucleic acids and protein found in the
nucleus, carrying genetic information).
Mostly, every type of cell that exists is categorized either as a prokaryotic or eukaryotic cell
based on the absence or presence of the nucleus within its cell. In summary, nucleus has two
primary functions:
It is responsible for storing the cell’s hereditary material or the DNA.
It is responsible for coordinating many of the important cellular activities such as protein
synthesis, cell division, growth and a host of other important functions.
4: Ribosomes (80S) - present in plant and animal cells
-Ribosomes are the organelle responsible for protein synthesis of the cell.
-They are found in the cell cytoplasm in large numbers and a few of them called functional
ribosomes can be found in the nucleus, mitochondria, and the cell chloroplast.
-Ribosomes are made up of ribosomal RNA (rRNA) and cell proteins and are manufactured
in the nucleolus of the nucleus.
-Ribosomes can be free in the cytoplasm, or (more commonly) are attached to the rough
endoplasmic reticulum. They are often found in groups called polyribosomes or polysomes.
- The main functions of ribosomes in plant cells:
Ribosomes major functions is to synthesize proteins for the cellular functions such as
cell repair mechanism.
They are also used in protein assembling and folding.
5: Endoplasmic reticulum (ER) - present in plant and animal cells
-The ER is a continuous network of folded membranous sacs housed in the cell cytoplasm.
-It is a complex organelle taking up a sizable part of the cell’s cytoplasm.
-There are two types of ER based on their structure and the function they perform. This
include rough endoplasmic reticulum (RER) and the smooth endoplasmic reticulum
(SER). RER- they have ribosomes attached to their surface membrane; SER - they
lack ribosomal attachment.
-The endoplasmic reticulum known for its high dynamics functions in eukaryotic cells;
playing major roles in synthesizing, processing, transporting and storing proteins, lipids, and
chemical elements.
- Summary of functions of endoplasmic reticulum:
RER is involved in synthesis of proteins, which are transported from the cell to the
Golgi bodies, which carry them to other parts of the plant to help in its growth.
-
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SER plays a role in synthesis, secretion and storage of lipids, metabolism of
carbohydrates and manufacturing of new membranes. This is enhanced by the
presence of several enzymes bound to its surface.
ER has been linked to the regulation of excess calcium levels in plant cells.
ER also act as plant sensors - mediate plant response to certain external stimuli e.g. light
intensity, temperature, and atmospheric pressure.
6: Golgi bodies (Apparatus) - present in plant and animal cells
-The Golgi bodies has multiple names such as Golgi complex or Golgi apparatus. The name
is after the scientist (Camillo Golgi) who discovered the organelle.
-Golgi bodies are found in all the eukaryotic cells, plants as well as animals.
They are complex membrane-bound organelle found in the cytoplasm (cytosol) of eukaryotic
cells.
[Golgi bodies are series of flattened membrane vesicles (sac-like structures) formed from the
endoplasmic reticulum (they lie just next to the endoplasmic reticulum and near the nucleus)].
-The Golgi apparatus is the processing, packaging and secreting organelle of the cell, so it
is much more common in glandular cells. They work closely with the SER, to modify
proteins for export by the cell.
Their main function of Golgi bodies is transportation of proteins from RER to the cell
membrane for export to various destinations, such as lysosomes, plasma membrane or
secretion.
They also take part in the transport of lipids and the formation of lysosomes.
Golgi apparatus is the site for the synthesis of various glycolipids, sphingomyelin, etc.
Golgi apparatus also synthesize complex polysaccharides that are used in making plant
cell wall.
7: Lysosomes of the plant cell - present in plant and animal cells
-Lysosomes are sphere-shaped sacs (vesicles) filled with hydrolytic enzymes that have the
capability to break down many types of biomolecules (intracellular and extracellular
breakdown of substances).
-In other words, lysosomes are membranous organelles whose specific function is to
breakdown cellular wastes and debris by engulfing it with hydrolytic enzymes.
-Hydrolytic enzymes and other cellular debris or wastes are contained within the lumen of
lysosomes.
-Lysosomes work as the waste discarding structures of the cell by processing undesirable
materials and degrading them, both from the exterior of the cell and waste constituents inside
the cell.
-Due to their peculiar function, lysosomes are also known as the “suicide bags” of the cell.
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-But sometimes, the digestive enzymes may end up damaging the lysosomes themselves, and
this can cause the cell to die; a process known as autolysis. Hence, lysosomes are known as
“Suicidal Bags” of the cell.
- In summary, lysosomes serve two functions:
Intracellular digestion of food substances required by the cell as well as removal of waste
products.
Autolytic action : cell parts (e.g. cell organelles) that should be gotten rid of are destroyed
by the action of lysosomal enzymes.
8: Vacuoles - present in plant and animal cells
-Vacuoles are membrane-bound cell organelles present in the cytoplasm and filled with a
watery fluid containing various substances.
-The term “vacuole” means “empty space”. They help in the storage and disposal of various
substances. They can store food or other nutrients required by a cell to survive. They also
store waste products and prevent the entire cell from contamination.
-The vacuoles of some plants contain poisons (e.g. tannins) that discourage animals from
eating their tissues.
Some unicellular organisms have feeding vacuoles for digesting food or contractile vacuoles
for expelling water (osmoregulation).
The vacuoles in plant cells are larger than those in the animal cells.
- In summary, the main functions of vacuole include:
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Storage - a vacuole stores sugars, salts, minerals, pigments, lipids and proteins within the
cell. The solution that fills a vacuole is known as the cell sap. The vacuole is also filled with
protons from the cytosol that helps in maintaining an acidic environment within the cell.
Turgor Pressure (cell turgidity) - the vacuoles are completely filled with water and exert
force on the cell wall. This is known as turgor pressure. It provides shape to the cell and
helps it to withstand extreme conditions.
9: Cytoskeleton - present in plant and animal cells
-Just as your body depends on your skeleton to maintain its shape and size, so a cell needs
structures to maintain its shape and size.
-In animal cells, which have no cell wall, an internal framework called the cytoskeleton
maintains the shape of the cell, and helps the cell to move.
-Thus, cytoskeleton is a network of long, thin protein fibers that provide an anchor for
organelles inside the cell. The cell’s shape and movement depend on the cytoskeleton. -
There are two types of protein fibers make up the cytoskeleton:
Microfilaments (contractile) - they are made of actin, and are common in motile cells - they
help give the cell shape and enable the entire cell or parts of the cell to move.
Microtubules (rigid, hollow tubes - made of tubulin) – they form a firm skeleton for the
cell. They assist in moving substances within the cell.
- In summary, functions of the cytoskeleton include:
Mitotic cell division (i.e. division of the cell cytoplasm by a mechanism known as
cytokinesis, forming two daughter cells);
Cytoplasmic streaming - a process of cytosol (cytoplasm) flow all over the cell, transporting
nutrients and cell organelles;
Plays a role in maintaining the cell shape, structural support and retain tension within the
cell; Transportation of cellular molecules (materials) within the cell using microtubules.
Used in formation of plant cell wall.
Cell signaling activities.
10: Cilia and Flagella - present in plant and animal cells
-Cilia and flagella are structures that project from the cell, where they assist in movement.
-Cilia (singular: cilium) are short, and numerous and hair-like.
-Flagella (singular: flagellum) are much longer, fewer, and are whip-like.
-Protista commonly use cilia and flagella to move through water.
-Sperm use flagella (many, all fused together) to swim to the egg.
-Cilia line our trachea and bronchi, moving dust particles and bacteria away from the lungs.
11: Cell wall - present in plant cells only
-It is the rigid outer cover of the plant cell (made of cellulose) with a major role of protecting the
plant cell; giving the plant cell its shape.
-The primary role of the cell wall is defined to be a mechanical and structural function, that is
highly effective in serving the plant cell.
-A part from cellulose, plant cell walls are also made of pectin, lignin and other polysaccharides.
-Fungi such as mushrooms and yeast also have cell walls which are made of chitin.
-Animal cells do not have a cell wall.
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- In summary, functions of the plant cell wall include:
Providing the cell with mechanical protection and shielding the cell from the chemically
harsh environment.
It is semipermeable hence it allows in and out, the circulation of materials such as water,
molecular nutrients, and minerals.
It provides a rigid building block to stabilize the plant to produce some of its structures, for
example, the stem and leaves of the plants.
It provides a site for storage of some elements such as the regulatory molecules that detect
pathogens in the plant, hindering the development of diseased tissue.
The cellulose fibers serve as structural and supportive functional layers when the cell
vacuoles are filled with water, exerting turgor pressure on the cell wall, thus maintaining the
plants’ stiffness and preventing plants from losing water and withering.
12: Plastids - present in plant cells only
-Plastids are double-membrane organelles which are found in the cells of plants and algae.
-They have characteristic pigments that aid their role of food processing (manufacture) and
storage in plants and algae. These pigments also determine the colour of the plant (cell).
- Summary of functions of plastids:
They are actively involved in manufacturing food for the plant by photosynthesis due to the
presence of chlorophyll pigment in the chloroplast.
They also store food in the form of starch.
They have the ability to synthesize fatty acids and terpenes that produces energy for cellular
activities.
Palmitic acid, a component synthesized by chloroplasts is used in manufacturing the plant
cuticle and waxy materials.
Types of plastids
-Plastids are classified based on their functions and the presence of the characteristic pigments.
They include:
Chloroplasts - green plastids used in photosynthesis;
Chromoplasts - coloured plastids used to synthesize and store plant pigments;
Gerontoplasts - they dismantle photosynthetic apparatus during aging of plants;
Leucoplasts - they are colourless plastids used to manufacture terpene substance that
protects the plants.
13: Chloroplast - present in plant cells only
-These are organelles found in all green plant cells and algal cells. They are the food producers
of plants. Chloroplasts are found in the guard cells located in the leaves of the plants. They
contain a high concentration of chlorophyll that traps sunlight. Chloroplasts are not present in
animal cells.
-They are oval- or biconcave in shape.
-Chloroplast has its own DNA and can reproduce independently, from the rest of the cell . They
also produce amino acids and lipids required for the production of chloroplast membrane.
-Each chloroplast encloses a system of flattened, membranous sacs called thylakoids, which
contain chlorophyll. The thylakoids are arranged in stacks called grana.
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- The main functions of chloroplasts include:
Photosynthesis: - are sites of food synthesis for plant cells through photosynthesis; Plant
colour: - they give plants their green colour
14: Mitochondria - present in animal and plant cells
-Mitochondria (singular: mitochondrion) are membrane-bound organelles present in the
cytoplasm of all eukaryotic cells, that produces adenosine triphosphate (ATP), the main
energy molecule used by the cell.
-They are the sites of aerobic respiration, in which energy from organic compounds is
transferred to ATP. For this reason they are sometimes referred to as the ‘powerhouse’ of the
cell.
-Mitochondria are relatively large organelles (second only to the nucleus and chloroplasts).
-There are hundreds of mitochondria within a single plant and animal cell.
-Mitochondria are numerous in cells that have a high energy requirement - e.g. phloem pigment
of the plant cell, and the neighboring cells that have high metabolism rates. This is to supply
energy that support various cellular activities, like the transportation of food through the sieve
tubes. Also, our muscle cells contain a large number of mitochondria, as do liver, heart and
sperm cells.
- Functions of mitochondria:
Production of energy for cellular activities.
Mitochondria also regulates the metabolic activity of the cell; It promotes the growth of
new cells and cell multiplication.
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TOPIC: 2 MICROBIAL
NUTRITION:
-Microorganisms (in this case bacteria), like all cells, require nutrients for the maintenance of
their metabolism and for cell division.
-Microbial nutrient can be divided into those that are required in large quantities
(macronutrients, major elements or macroelements); those which are needed in small
quantities (micronutrients, microelements) and those that required in very low concentrations
(in traces or trace amounts / trace elements).
Macronutrients / Macroelements:
-Major nutrients required in large quantities include:
i. Carbon: forms the structural backbone or central component of for organic molecules
e.g.
proteins, carbohydrates, nucleic acids and lipids. Carbon may be obtained from organic
e.g. sugars or inorganic sources (see Table below). Some organisms can derive carbon
from carbon dioxide (CO2).
ii. Hydrogen: also a constituent of cellular water (H2O) and organic compounds. Hydrogen
is required by some microorganisms to reduce carbon dioxide in the synthesis of
macromolecules.
iii. Oxygen: a constituent of cellular water and organic compound e.g. glucose, nucleic
acids. Oxygen is also used in aerobic respiration of many microorganisms and is
obtained from water.
iv. Nitrogen: needed for the synthesis of proteins (amino acids) and nucleic acids, as well
as for important molecules such as adenosine triphosphate (ATP.) Some microorganisms
get nitrogen by assimilating (‘fixing’) gaseous nitrogen (N2) while others require all 20
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amino acids to be provided in growth medium. Other species of microbes can assimilate
nitrogen
from an inorganic source such as nitrate, while others can utilize ammonium salts or
urea as source of nitrogen.
v. Phosphorus: occurs in nucleic acids and in membranes (phospholipids); also a
component of high energy compounds e.g. adenosine triphosphate (ATP), adenosine
diphosphate (ADP) and nicotinamide adenine dinucleotide phosphate (NADP).
Phosphorus is generally provided as inorganic phosphate ions, often as a pH buffer.
vi. Sulphur: required for synthesis of Sulphur containing amino acid (methionine, cysteine)
and coenzymes or cofactors (vitamins) that are used in cellular respiration and
photosynthesis. Sulphur is often supplied as an inorganic sulphate or sulphide salt
Micronutrients / Microelements:
-Minor nutrients required in small quantities include:
i. Potassium: maintain correct osmotic pressure and electric potential of a cell; also
involved in normal cell division and synthesis of proteins, as well as a counter-ion for
the DNA phosphate backbone synthesis.
ii. Magnesium: Magnesium ions are involved in the stabilization of ribosomes, and some
are necessary for maintaining cell wall and membrane integrity. Magnesium ions also
act as a co-factor for enzymatic reactions.
iii. Sodium: are used in chemiosmotic energy pumps.
iv. Calcium: makes endospore resistant, also a co-factor in some enzymes
v. Iron: a constituent of photosynthetic pigments (chlorophyll) in cyanobacteria; also acts
co-factor for enzymatic reactions. Iron is also used by some bacteria to make toxins e.g.
Corynebacterium diphtheriae which causes diphtheria.
Trace elements:
-Chemical required in very low concentrations (i.e. required in traces) include:
i. Manganese: a co-factor for microbial enzymatic reactions. ii.
Molybdenum: a co-factor for microbial enzymatic reactions.
iii. Cobalt: a constituent of some vitamins e.g. vitamin B12 (cyanocobalamin).
iv. Silicon: a constituent of microbial cell walls and cell membrane.
Others:
i. Growth factors: like amino acids for synthesis of proteins; purines and pyrimidines
(required for nucleic acid synthesis).
ii. Vitamins: synthesis of enzymes and coenzymes.
iii. Sterols: stabilize cell membrane in bacterial species like Mycoplasmas which lack cell
walls.
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Nutritional Categories of Microorganisms:
-Microorganisms can be categorized according to how they obtain their carbon and energy.
Carbon is the most abundant component of the microbial cell, and most microorganisms obtain
their carbon in the form of organic molecules, derived directly or indirectly from other
organisms.
-Microbial carbon nutrition is divided into two classes: heterotrophs and autotrophs.
Heterotrophs: use one or more organic compounds (e.g. hydrocarbons, lipids, organic acids,
simple sugars and polysaccharides) as their source of carbon.
Autotrophs: utilize carbon dioxide (CO2) as their sole or primary source of carbon.
-Autotrophs can further be divided into two groups: chemoautotrophs and photoautotrophs.
Chemoautotrophs (chemotrophs): obtain their energy as well as their carbon from
inorganic (chemical) compounds, and they build their complex molecules from carbon
dioxide.
Photoautotrophs (phototrophs): have photosynthetic pigments enabling them to convert
light energy into chemical energy. They use energy from sunlight, and carbon from carbon
dioxide.
-Heterotrophs also fall into two categories: chemoheterotrophs and photoheterotrophs.
Chemoheterotrophs: obtain energy as well as carbon from organic (chemical) compounds.
Photoheterotrophs: use sunlight as an energy source, but require an organic carbon source
(they cannot fix carbon dioxide into organic carbon).
-Microorganisms (both chemotrophs and phototrophs) can further be categorized into two
groups (i.e. lithotrophs and organotrophs) based on their sources of hydrogen atoms or
electrons to drive their energy-generating systems:
Lithotrophs: organisms that use inorganic molecules (such as water (H2O), hydrogen
sulphide (H2S) or ammonia (NH3) as a source of electrons or hydrogen.
Organotrophs: organisms that use organic molecules (such as sugars) to obtain electrons or
hydrogen.
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Nutrient Uptake: How do Nutrients Get into the Microbial Cell?
-Having found a source of a given nutrient, a microorganism must: Have some means of
taking it up from the environment; Possess the appropriate enzyme systems to utilize it.
-The plasma membrane represents a selective barrier, allowing into the cell only those
substances it is able to utilize. This selectivity is due in large part to the hydrophobic nature of
the lipid bilayer. A substance can be transported across the cell membrane in one of three ways,
known as simple diffusion, facilitated diffusion and active transport.
1. Simple diffusion:
-In simple diffusion, small molecules move across the membrane in response to a concentration
gradient (from high to low), until concentrations on either side of the membrane are in
equilibrium. The ability to do this depends on being small (H2O, Na+, Cl-) or soluble in the lipid
component of the membrane (e.g. non-polar gases such as O2 and CO2).
-The diffusion or movement of nutrients (or molecules) through the semipermeable membrane
doesn’t involve the assistance of specialized transporter or carrier proteins.
-There is no expenditure of cellular energy in simple diffusion.
2. Facilitated diffusion:
-Facilitated diffusion involves passive movement of larger polar molecules (nutrients e.g.
glucose and amino acids) across the cell by the help of membrane-spanning proteins,
also known as carrier proteins or permeases.
-Molecules along a concentration gradient (from high concentration low concentration), thus
does not require the expenditure of energy (ATP hydrolysis).
-The transporter or carrier proteins assist the molecules to cross the lipid bilayer membrane.
-The participation of carrier proteins in facilitated diffusion requires no direct input of energy. It
is driven solely by the concentration gradient across the membrane and is reversible.
3. Active transport:
-Active transport involves the movement of materials (molecules, nutrients) against a
concentration gradient (from low concentration high concentration).
-Since materials are moving against the gradient, it requires the expenditure of energy (e.g. ATP
hydrolysis).
-As in facilitated diffusion, active transport also involves specific transmembrane or carrier
proteins; many of which are highly specific, whereas others function with groups of related
compounds.
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TOPIC: 3
MICROBIAL GROWTH:
-When we consider growth as applied to a multicellular organism such as a tree, a fish or a
human being, we think in terms of an ordered increase in the size of an individual.
-Growth in unicellular microorganisms such as bacteria, yeasts and protozoans, however, is more
properly defined in terms of an increase in the size of a given population . This may be
expressed as an increase in either the number of individuals or the total amount of biomass.
-Microbial growth can be defined as an orderly increase in cellular components, resulting in cell
enlargement and eventually leading to cell division. This definition is not strictly accurate as it
implies that a consequence of growth is always an increase in cell numbers. However, under
certain conditions growth can occur without cell division, for example, when cells are
synthesizing storage compounds, e.g. glycogen. In this situation the cell numbers remain
constant, but the concentration of biomass continues to increase. This is also true for some
species fungi, that do not divide into separate cells. Their growth results only in increased size.
-Thus, growth of a cell is a complex process involving:
1. Entrance of basic nutrients into the cell;
2. Conversion of these compounds into energy and vital cell constituents;
3. Replication of the chromosome;
4. Increase in size and mass of the cell;
5. Division of the cell into two daughter cells, each containing a copy of the genome and other
vital components.
- Bacterial growth involves both an increase in the size of individuals and increase in the
number of individuals. Whatever the balance between these two processes, the net effect is an
increase in the total mass (biomass).
Biomass is the total amount of cellular material in a system.
-Bacteria divide by binary fission where individual cells enlarge and divide to yield two
progenies (or two identical daughter cells) of approximately equal size (Fig. 4.1). By identical
means, two cells divide into four, four into eight and so on, leading to an exponential increase
in cell numbers. Exponential growth—is defined as increase in growth by a power of 2:
-Each time a cell divides is called a generation and the time taken for the cell to divide (and its
population to double) is referred to as the generation time. Therefore, the generation time or
doubling time (td) is the time required for a microbial population to double. Theoretically, after
one generation, both the microbial cell population and biomass concentration have doubled. For
example, under favorable conditions, E. coli takes approximately 20 minutes to double its
population and Mycobacterium takes 24 hours.
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Bacterial Growth Curve:
-When a few bacteria are inoculated into a liquid growth medium and the population is counted
at intervals, it is possible to plot a bacterial growth curve that shows the growth of cells over
time.
-The bacterial growth curve can be divided into four major phases: (i) lag phase (ii)
exponential or log (logarithmic) phase (iii) stationary phase, and (iv) decline (death)phase.
These phases reflect the physiologic state of the organisms in the culture at that particular time.
Inoculum is the term given to the cells used to ‘seed’ a new culture.
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1): Lag phase
-This is the period of time that elapses between the inoculation of the culture medium and
detection of any visible growth.
-In most bacterial growth, the phase takes approximately 4 hours.
Events that occur during lag phase:
1. During this period, the bacterial culture is adapting to the new growth medium.
2. The number of cells does not increase.
3. The cells are actively metabolizing the necessary enzymes and growth factor (e.g. amino
acids, vitamins or cofactors etc.).
4. The cells increase in size only but do not divide.
NB: The length of lag phase depends on the bacterial species, nature of the medium
(nutrient), size of inoculum and environmental factors such as pH and temperature. If these
conditions are optimum, the cells quickly shift to the log phase
2): Logarithmic (Log) / exponential phase
-Once the culture has adjusted during the lag phase, it enters log phase where growth is noticed.
Events that occur during log phase:
1. Cells multiply exponentially and the population increases.
2. Cells have shortest generation time.
3. All the cells are metabolically similar.
4. The rate of utilization of nutrients and excretion of waste is also exponential.
5. Reproduction is greater than death.
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6. The culture undergoes a balanced growth (increase in biomass is accompanied by
comparable or orderly increase in cellular components e.g. proteins, DNA, RNA, enzymes).
NB: Exponential phase is of limited duration because of (i) exhaustion of nutrients; (ii)
accumulation of toxic metabolic end products; (iii) rise in cell density, (iv) change in pH;
and (v) decrease in oxygen tension (in case of aerobic organisms).
3): Stationary phase
-During this phase there is no further increase in bacterial cell numbers. The growth rate is
exactly equal to the death rate.
Events that occur during stationary phase:
1. The nutrients became a limiting factor / are depleted or exhausted.
2. Waste secretion increase and becomes too much for cells to bear; accumulation of toxic or
inhibitory end products.
3. Change in physical conditions of bioreactors or culture vessels.
4. The rate of reproduction equals death rate and the population remains fairly constant.
4): Decline phase
-The death phase starts when the number of viable bacterial cells begins to decline. In other
words, the number of deaths eventually exceeds the number of new cells formed, and the
population enters the death phase, or logarithmic decline phase.
Events that occur during decline phase:
1. Toxins / waste accumulations is exponential and poisons the cells.
2. There is production of self-digesting enzymes / lytic enzymes.
3. Cells die exponentially, there is less reproduction.
NB: Some bacterial cells survive the phase by the vegetative cells transforming into
endospore e.g. Bacillus subtilis
Batch Culture and Continuous Culture:
-Microbial growth, cultivation or fermentations in liquid media can be carried out under different
operating conditions, i.e. batch growth and continuous growth.
1. Batch Culture System :
-Batch culture system, also known as batch growth involves a closed system where appropriate
nutrients and other conditions are provided for growth, then an inoculum is added and the
culture incubated. No further nutrients are added and no waste products are removed, thus
conditions in the culture are continually changing. This results in active growth being of limited
duration.
-In other words, batch cultures involve growing bacterial cells in limited volume of growth
medium in a culture flask, tube or agar plate.
-The four (4) phases of bacterial growth described above (i.e. lag phase, log phase, stationary
phase and decline phase) apply to a batch culture.
2. Continuous Culture System:
-Continuous culture system, also known as continuous fermentation or continuous growth is
an open system where nutrient concentrations and other conditions are held constant, and the
cells are held in a state of exponential growth. This is achieved by continuously adding fresh
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culture medium and removing equal volumes of the old or waste products. Parameters such as
pH can also be monitored and adjusted. The equipment used to do perform continuous growth is
called a chemostat.
-A continuous growth system can be improved by using a turbidostat, which is a device with a
light sensing component (known as a photocell). The photocell measures the absorbance or
turbidity of cells and regulates or adjusts flow rate of the culture medium automatically—thus
maintaining a predetermined turbidity or cell density.
Applications of Continuous Culture Fermentations:
-Useful in performing studies on all aspects of cell growth, physiology and biochemistry; -
Useful for ecological studies—how microbes respond to environmental conditions;
-Useful as a genetics tool for the examination of mutation rates, mutagenic effects etc.
-Useful in industrial fermentations—e.g.—production of alcohol and antibiotics etc.
Factors Affecting (influencing) Microbial Growth:
1. Nutrition :
-All living organisms (microorganisms) have certain basic nutritional requirements: sources of
carbon, nitrogen, energy, and essential growth factors (amino acids, minerals and vitamins etc.)
are needed to support growth. Adequate supply of macronutrients, micronutrients, trace
elements and growth factors must be provided for optimal microbial (bacterial) growth.
2. Osmotic pressure :
- Microorganisms obtain almost all their nutrients in solution from the surrounding water.
Thus, they require water for growth, and their composition is 80–90% water. If the
bacteria are grown in hypertonic environment (i.e. higher osmotic pressure than
cellular contents), water inside the cell will start to move outside the cell and the
bacterial cell will shrink and will die, a process known as, plasmolysis. If the bacteria
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are grown in hypotonic environment (lower osmotic pressure than cellular contents),
water will start
to move inside the cell and bacterial cell will swell and burst, a process known as
plasmoptysis. Due to their rigid cell walls, bacteria have become more tolerant to
variations in osmotic pressures. Thus, different bacteria live in different osmotic
pressure.
-Osmotic pressure: is the pressure exerted on bacterial cell surface as a result of
difference in solute concentration between the inside and outside of a cell.
3. Temperature :
- Each bacterial species has an optimal temperature for growth and a temperature range
above and below which growth is blocked. The temperature at which growth occur best
is known as the ‘optimum temperature’. Thus, bacteria pathogenic for humans usually
grow at 37ºC (our body temperature).
- Bacteria are divided into three groups on the basis of temperature ranges through which
they grow:
a. Mesophiles ( moderate temperature loving ) :
- Mesophiles (mesophilic) are microorganisms that grow best at intermediate
(moderate) temperatures between 20 °C and 45 °C (neither too hot nor too cold),
with an optimum growth temperature in the range of 30–39°C.
- Most human pathogens have optimum growth temperatures somewhere between
30°C and 40°C (human body temperature is 37°C).
- Examples: Streptococcus pyogenes, Streptococcus pneumoniae, Staphylococcus
aureus, Salmonella sp., Proteus vulgaris, and specific strains of Escherichia coli.
b. Psychrophyles ( cold loving ) :
- Psychrophiles (psychrophilic) are cold-loving, cold-tolerant (cold-adapted)
microorganisms that are capable of growth and reproduction in low
temperatures, ranging from −20°C to 20°C. They are found in places that are
permanently cold, such as the polar regions and the deep sea.
- Psychrophiles are soil and water saprophytes and though not of direct medical
importance, may cause spoilage of refrigerated food. These organisms may be
capable of growth in food and pharmaceuticals stored at normal refrigeration
temperatures (0-8ºC).
- Examples: Listeria monocytogenes, Aeromonas hydrophila, Clostridium
botulinum, and Yersinia enterocolitica.
c. Thermophiles ( heat-loving ) :
- Thermophiles (thermophilic) are microbes that can thrive in temperatures as high
as 55°C (minimum temperature: 45°C, optimal temperature range: 55–65°C,
maximum temperature: 80°C).
- Thermophiles can be found in various geothermally heated areas of the earth,
including soil and water associated with volcanic activity and in habitats directly
exposed to the sun e.g. deep sea hydrothermal vents, hot springs, geysers etc.
- They may cause spoilage of under processed canned food and can be a source of
enzymes with remarkable thermotolerant properties such as Taq polymerase
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(isolated from Thermus Aquaticus), a key enzyme used in the polymerase chain
reaction (PCR).
- Microbes that can tolerate temperatures in excess of 100°C (with 80°C being
ideal) are known as hyper-thermophiles (or extreme thermophiles).
- Thermophiles include Bacillus stearothermophilus, Thermoplasma acidophilum,
Thermus aquaticus, etc., whereas extreme thermophiles include Pyrodictium
occultum and Pyrococcus abyssi.
4. Hydrogen ion concentration (pH) :
- Microbial growth and survival is also influenced by the pH of the habitat. The pH is
defined as the degree of acidity or alkalinity (basicity) of a solution. It is expressed by
the pH scale, a series if numbers ranging from 1 to 14. As the pH value decreases toward
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0, the acidity increases and as the pH increases toward 14, the alkalinity increases.
pH<7 is acidic. pH =7 is neutral. pH>7 is alkaline.
-Neutrophilic (neutrophiles) bacteria grow best at near neutral pH value (e.g. Escherichia
coli, Staphylococcus aureus and Salmonella enterica etc.).
-Acidophilic (acidophiles) bacteria prefer to grow at low pH value (acidic medium) e.g.
Thiobacillus acidophilus, Lactobacillus acidophilus, Acetobacter aceti, Helicobacter
pylori etc.
-Alkaliphilic (alkaliphiles) bacteria prefer to grow at high pH value (alkaline medium)
e.g. Pseudomonas alcaliphila, Bacillus pseudofirmus, Bacillus lehensis, Vibrio cholerae
etc.
- Most bacteria can live and multiply within the range of pH 5 (acidic) to pH 8 (basic) and
have a pH optimum near neutral (pH 7); high pH (basic) or low pH (acidic) habitats can
damage enzymes and other cellular substances.
- Most pathogenic bacteria grow best at a neutral or slightly alkaline pH (7.2 to 7.6) while
Most fungi grow well at lower pH (pH 4 or 5) which is acidic.
5. Oxygen :
- Microorganisms can be classified into five main groups on the basis of their
requirements for oxygen:
a. Obligate aerobes: grow only in the presence of oxygen.
b. Obligate anaerobes cannot tolerate oxygen at all; exposure to it results in their
death.
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c. Facultative anaerobes are able to grow in the presence of oxygen, but can also
survive in the absence of oxygen. Undergo both aerobic and anaerobic growth;
greater growth in presence of oxygen.
d. Aerotolerant anaerobes are basically anaerobic (only anaerobic growth); but
continues to grow in presence of oxygen—since they don’t utilize it.
e. Microaerophiles require oxygen for growth, but only in low concentrations (i.e.
only aerobic growth; oxygen required in low concentration)—higher concentrations
harmful.
6. Carbon dioxide :
- All bacteria require small amount of carbon dioxide as a source of carbon for their
growth. Thus, this requirement is usually met by the carbon dioxide present in the
atmosphere, or produced endogenously by cellular metabolism. The microbes can also
be cultured in growth media containing bicarbonate (HCO−3) as carbon source or an be
incubated in a CO2-enriched atmosphere. Microorganisms that thrive in the presence of
high concentrations of carbon dioxide are known as capnophiles.
- Examples: Brucella abortus, Campylobacter jejuni, Helicobacter pylori, Neisseria
gonorrhoeae, Haemophilus influenza, Streptococcus pneumoniae etc.
7. Salinity :
- Salt content of the medium affects bacterial growth.
- Bacteria that grow best in high salt concentrations are known as halophiles (halophilic
bacteria).
- Moderate halophiles require 3% salt concentration.
- Extreme halophiles require 15% salt concentration.
- Most bacteria cannot tolerate high salt concentration. High salt concentration disrupts
membrane transport systems and denatures proteins of bacteria but halophiles have
adaptive mechanisms to tolerate high salt concentration.
8. Light / radiation :
- Phototrophic organisms require light in order to carry out photosynthesis. Therefore,
when growing microbes in the laboratory, care must be taken that simple light of the
correct wavelength (of 600 nm) is used, and that the source used does not also act as a
heat source.
- Fluorescent light produces little heat, but does not provide the wavelengths (of approx.
750 nm) needed by purple and green photosynthetic bacteria.
- UV light is bad for the growth of bacteria.; it changes the structure of DNA and cause
mutation. UV light will make loops in DNA thus interfering with process of replication
and transcription.
- Bacteria should not be exposed to ionizing radiations such as gamma (γ)—which can
cause breakage of DNA strands.
9. Hydrostatic pressure :
- Hydrostatic pressure: is the pressure exerted on microorganism (bacteria) by the weight
of a water column.
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- Microorganisms that grow best in conditions of high atmospheric pressure are known as
barophiles (barophilic microbes).
- In nature, many microorganisms are never subjected to pressures in excess of
1atmosphere (atm) and higher pressures generally inhibit microbial growth by
inactivating enzymes and disrupting membrane transport process. However, many
marine organisms must be barotolerant, as they live at depths in oceans where they
may be subjected to pressures of up to several hundred atmospheres, and only fail to
grow at above 200–600atm. Some may even grow better under these conditions and are
truly barophilic, capable of living under 700–1000atm.
10. Water (moisture) :
- The most important element for bacterial growth is water.
- The rate of metabolic and physiological activities of bacteria is determined by the
amount of water available in the culture media.
- Water dissolves sugar, salts, and other chemicals, making them accessible to bacteria.
11. Surface :
- Microorganisms does not grow in air they need some medium for their growth. These
medium may be liquid or solid.
- In liquid medium, bacteria can grow at a very rapid rate up to 1012-14, and if the water is
mixed regularly or agitated they can reach up to 1016-20. This is because in liquid
medium, nutrients are easily diffusible and easily available.
- In solid medium, bacterial growth rate is slow as compared to liquid medium. However,
if the nutrients are provided is solid medium, bacteria can grow up to 106-8. Most
commonly used solid media is “agar”.
12. Mechanical and sonic stresses :
- In spite of rigid walls of bacteria, they may be ruptured by mechanical stress such as
grinding or vigorous shaking with glass beads. Exposure to ultrasonic vibration may
also disintegrate bacteria.
TOPIC: 4
HISTORICAL PERSPECTIVES ON MICROBIAL GENETICS:
-Define the following terminologies:
Genetics: is the study of how genes carry information, how they are replicated and passed
to other generations, and how they affect the characteristics of an organism.
Microbial genetics means genetics of microbes (bacteria, archaea, viruses, including
bacterial viruses i.e., bacteriophages and unicellular or mycelial eukaryotes including yeasts,
fungi, algae and protozoa).
The following scientists contributed greatly to the development of Microbial Genetics:
- Walter Sutton and Theodore Boveri (1902-1903): the two German scientists developed the
Boveri–Sutton chromosome theory, also known as the chromosome theory of inheritance or
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the Sutton Boveri theory; a fundamental unifying theory of genetics which identifies
chromosomes as the carriers of genetic material.
- William Bateson: An English biologist who was the first person to use the term “genetics” to
describe the study of heredity i.e. he coined the term genetics.
- Frederick Griffith: British bacteriologist whose 1928 experiment with bacterium
(Streptococcus pneumoniae) was the first to reveal the “transforming principle,” which
showed how healthy cells could transform into virulent, disease-causing, cells. Griffith's
experiments demonstrated the concept of bacterial transformation, whereby a bacterium
distinctly changes its form and function.
- Oswald T. Avery, Colin M. MacLeod, and Maclyn McCarty: Based at the Rockefeller
Institute in New York, their study in 1944 (known as Avery–MacLeod–McCarty experiments)
gave molecular explanation to Griffith’s experiments for bacterial transformation of
Streptococcus pneumoniae—by demonstrating that that DNA is the substance that causes
bacterial transformation i.e. DNA acts as the carrier of genetic information.
- Rosalind Franklin and Maurice Wilkins: While working at King’s College in London in
1950, they used X-ray crystallographic equipment (X-ray imaging) to determine the three
dimensional structure of DNA. Their work laid foundation for James Watson and Francis Crick
to suggest that DNA is a double-helix polymer in 1953.
- Alfred Hershey and Martha Chase: In 1952, performed Hershey–Chase experiments to
confirm that DNA was/is a genetic material; an idea that had first been demonstrated in the
1944 by Avery–MacLeod–McCarty experiments.
- George W. Beadle (1903-1989) and Edward L. Tatum (1909-1975): They investigated
genetics of tryptophan metabolism and nicotinic acid synthesis in the fungus (bread mold)
Neurospora crassa. They proposed a direct link between genes and enzymatic reactions, known
as the “one gene-one enzyme hypothesis”. The one gene, one enzyme hypothesis entails the
idea that each gene encodes a single enzyme. However, today, we know that this idea is
generally (but not exactly) correct. Later this concept was broadened to one gene–one
polypeptide, because a large class of structural genes exists in which each gene encodes a
single polypeptide, which may function either independently or as a subunit of a more complex
protein.
- Joshua Lederberg and Edward Tatum (1946): He demonstrated the exchange of genetic
factors through conjugation in Escherichia coli, mediated by plasmids (“fertility factors”).
Later, processes such as transformation, transduction and chromosomal gene mobilization led to
genome (chromosome) mapping in bacteria.
- François Jacob (1920–) and Jacques Monod (1910-1976): French biologists who formulated
the operon model. Based on studies on the induction of enzymes of lactose catabolism in the
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bacterium Escherichia coli, the operon has provided the groundwork for studies on gene
expression and regulation.
TOPIC: 5
STRUCTURE AND FUNCTIONS OF THE GENETIC MATERIAL:
Nucleic Acid Structure:
-A substance called deoxyribonucleic acid ( DNA ) is the substance of which genes are made.
DNA and another substance called ribonucleic acid ( RNA ), are together referred to as nucleic
acids because they were first discovered in the nuclei of cells.
- Nucleotides: Nucleotides are the structural units of nucleic acids. Nucleotides are named
according to their nitrogenous bases.
- Parts of Nucleotide:
Each nucleotide has three parts:
a. A nitrogen-containing base
Purines and pyrimidines: The nitrogen-containing bases are cyclic compounds made
up of carbon, hydrogen, oxygen, and nitrogen atoms. The bases are named adenine
(A), thymine (T), cytosine (C), guanine (G), and uracil (U). A and G are double-
ring structures called purines, whereas T, C, and U are single ring structures
referred to as pyrimidines.
b. A pentose (five-carbon) sugar called deoxyribose or ribose.
c. A phosphate group (phosphoric acid).
- Nucleoside: The term nucleoside refers to the combination of a purine or pyrimidine plus a
pentose sugar; it does not contain a phosphate group
A: Deoxyribonucleic Acid (DNA) Structure:
- Double helix: According to the model proposed by Watson and Crick, a DNA molecule consists
of two long strands wrapped around each other to form a double helix (Fig. 10.1). The double
helix looks like a twisted ladder, and each strand is composed of many nucleotides. The two
strands are held together by weak hydrogen bonds between the nitrogenous bases of the
opposing strands.
- Sugar-phosphate backbone: Every strand of DNA composing the double helix has a
“backbone” consisting of alternating deoxyribose sugar and phosphate groups. The
deoxyribose of one nucleotide is joined to the phosphate group of the next. The nitrogen
containing bases make up the rungs of the ladder. Purine A is always paired with the
pyrimidine T and that the purine G is always paired with the pyrimidine C. The bases are
held together by hydrogen bonds; A and T are held by two hydrogen bonds, and G and C are
held by three (Fig.
10.2).
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- Base pairing: The characteristic bonding of A to T and G to C is called base pairing and is
fundamental to the remarkable functionality of DNA. Because of the rules of base pairing, one
strand can always be used as a template for the synthesis of the complementary opposing strand.
- Complementary: Because the sequence of bases of one strand is determined by the sequence
of bases of the other, the bases are said to be complementary. While the two strands of DNA in
the double helix are complementary, they are also antiparallel. That is, they are oriented in
opposite directions. One strand is oriented in the 5' to 3' direction and its complement is
oriented in the 3' to 5' direction. This also has important implications in the function and
synthesis of nucleic acids.
Code and Codon:
-Genetic information is stored in DNA as a code. The unit of code is known as codon. Therefore,
a codon consists of a sequence of three bases; which codes for a single amino acid. Thus, code
is triplet. Each codon specifies or codes for a single amino acid, but more than one codon may
exist for a single amino acid. Therefore, code is degenerate. For instance, the codons (triplets)
AGA, AGG, CGU, CGC, CGA and CGG all code for arginine. There are 64 codons, of which 3
(UAA, UAG and UGA) are nonsense codons—they don’t code for any amino acid, but act as
stop codons—i.e.—they act as punctuation marks, terminating the message for synthesis of a
polypeptide or a protein.
-There are specific codons which code for start and stop sequences. The start codon ( AUG )
indicates the beginning of the sequence to be translated, and the stop codons ( UAA , UGA ,
UAG ) terminate the protein synthesis. With the exception of methionine, all amino acids are
coded for by more than one codon.
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Cistron or Gene:
-A segment of DNA carrying a number of codons specifying for a particular polypeptide
(protein) is known as cistron or gene. A large number of genes constitute a locus and a large
number of loci constitute cell genome. DNA can be compared with a book of information.
Letters represent nucleotides, words represent codons, sentences represent genes, paragraphs
represent loci and entire book as DNA molecule or cell genome.
-A DNA molecule consists of a large number of genes, each of which contains hundreds of
thousands of nucleotides. The bacterial chromosome consists of a double-stranded molecule of
DNA arranged in a circular form. When straightened, it is about 1,000 μm in length. The length
of DNA is usually expressed as kilobases (1 kb = 1,000 base-pairs). Bacterial DNA is about
4,000 kb and the human genome about 3 million kb long.
- Introns and Exons: In higher forms of life, several stretches of DNA that do not appear to
function as codons occur between the coding sequences of genes. These apparently useless
noncoding intrusions are called introns , while the stretches of coded genes are called exons i.e.
introns are non-coding sequences on a gene, while exons are coding sequences on a gene which
are translated into proteins. During transcription, the genome is copied in its entirety, both
introns and exons. The introns are then excised from the RNA copy before being translated by
the ribosomes into proteins. This process occurs in eukaryotes and is known as splicing; the
introns are removed from the primary transcript (RNA molecule) and the exons are joined to
form a continuous sequence that specifies a functional polypeptide or protein.
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-There are specific codons which code for start and stop sequences. The start codon ( AUG )
indicates the beginning of the sequence to be translated, and the stop codons ( UAA , UGA ,
UAG ) terminate the protein synthesis. With the exception of methionine, all amino acids are
coded for by more than one codon.
-There are three basic categories of bacterial genes : - Genes that code for proteins—
structural genes
-Genes that code for RNA (rRNA and tRNA)
-Genes that control gene expression—regulatory genes
B: Ribonucleic Acid (RNA) Structure:
- Differences between DNA and RNA: RNA, the second principal kind of nucleic acid, differs
from DNA in several respects (check the differences between DNA and RNA during your
revision time).
- Types of RNA: Three major kinds of RNAs have been identified in cells. These are classified
as messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA). Each type of
RNA has a specific role in protein synthesis.
i. Messenger RNA (mRNA): Most genes encode proteins and are transcribed into messenger
RNA (mRNA). These molecules are translated during protein synthesis. Alternatively,
the gene product can be a ribosomal RNA (rRNA) or transfer RNA (tRNA), each of
which plays a different but critical role in protein synthesis.
•mRNA is direct carrier of genetic information from the nucleus to the cytoplasm.
•Usually a molecule of mRNA contains information required for the synthesis of one
protein molecule.
•Genetic information is present in mRNA in the form of genetic code.
ii. Transfer RNA (tRNA): Serves as adaptor molecule in protein biosynthesis. It carries
amino acids to site of protein synthesis. There is at least one tRNA molecule to each of
20 amino acids required for protein synthesis.
iii. Ribosomal RNA (rRNA): Ribosomes, composed of rRNA and proteins, also are
central to translation and provide the site at which translation occurs.
•rRNAs are required for the formation of ribosomes.
•rRNAs are involved in initiation of protein synthesis.
Flow of Genetic Information:
-The flow of information from DNA to RNA to protein is often referred to as the central dogma
of molecular biology (DNA→RNA→polypeptide/protein). The central dogma of molecular
biology denotes that DNA carries all genetic information. The flow of genetic information
includes the replication of DNA to make more DNA, the transcription of the DNA into mRNA
and the translation of mRNA into proteins. Replication of DNA first involves the separation of
the two strands of DNA followed by synthesis of new identical DNA strand by enzymes called
DNA polymerases. The RNA strand is synthesized by enzymes called RNA polymerases. The
RNA sequence will be complementary to the DNA sequence. The mRNA strands are then
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guided to the ribosomes for protein translation. Amino acid residues are brought to the mRNA
strand on the ribosomes by transfer RNA (tRNA).
PROKARYOTIC (BACTERIAL) GENOMES:
-Genome: The complete genetic information (either DNA or, in some viruses, RNA) of an
organism, typically expressed in the number of base pairs (bp).
-Bacterial genome is the total collection of genes carried by a bacterium, both on its
chromosome and on its extrachromosomal genetic elements or non-chromosomal element i.e.
plasmids, if any.
-Genomes of prokaryotic and eukaryotic cells are exclusively DNA.
-Genomes of viruses contain DNA or/and RNA.
- Significance of Studying Prokaryotic/Bacterial Genomics :
-Bacterial genomics can give us a broader understanding of how a bacteria functions, a bacteria's
origin, and the role bacteria play in our world/life that we can't study by other means (i.e.
through obtaining their DNA from the environment and studying it).
-Of medical interest, bacterial genomics is also anticipated to play a significant role in speeding
up the development of better therapies and vaccines for controlling disease-causing bacteria.
-Bacterial genomics will also be the cornerstone of anticipated DNA-based diagnostic tools that
will hopefully enable medical professionals (doctors) make quicker, more accurate diagnoses of
infectious disease.
- Size of Bacterial Genomes :
-Bacterial genomes are generally smaller and less variant in size between species when
compared to genomes of eukaryotes. The size of bacterial genomes ranges from 139 kbp to
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13,000 kbp. The smallest bacterial genome identified so far is from Mycoplasma genitalium, an
obligate intracellular (sexually transmitted) pathogen with a genome size of 0.58 Mbp (580 Kbp
≈ 580,000 bp). [NB: 1 Kbp = 1000 bp; 1 Mbp = 1,000,000 bp]. Mycoplasma genitalium is
restricted to the intracellular niche (i.e. mucous epithelial cells of the urinary and genital tracts
in humans) because it lacks genes encoding enzymes required for amino acid biosynthesis and
the peptidoglycan cell wall, genes encoding TCA (tricarboxylic acid) cycle enzymes, and many
other biosynthetic genes required for synthesis of lipids, nucleotides, and vitamins etc.
-In contrast to such obligate intracellular bacteria, free-living bacteria must dedicate many genes
toward the biosynthesis and transport of nutrients and building blocks. The smallest free-living
organisms have a genome size over 1 Mbp. Currently, largest sequenced prokaryotic genome is
Streptomyces coelicolor, 8.7 Mbp.
-Due to their small genome size, bacteria have none or relatively small amounts of repetitive
“junk” DNA (non-coding DNA; introns), i.e. in prokaryotes, most of the genome (85-90%) is
non-repetitive DNA; which means the genome is mainly composed of “coding DNA (exons)”
while non-coding regions only take a small part of the genome. The genomes of bacteria are
compact—essentially all of their DNA is “functional” (i.e. contains genes or gene regulatory
elements).
-Most prokaryotes contain only one copy of their genes/chromosomes (i.e. they are haploids),
whereas eukaryotes usually have two distinct copies of each chromosome/gene (i.e. they are
diploids). With only one chromosome, alteration of a bacterial gene (mutation) will have a
more obvious effect on the cell.
- Gene Content :
-Gene portion of bacterial genome is around 85-95%. Bacteria possess few genes such as in case
of Mycobacterium genitalium (480 genes). The highest gene content is present in
Bradyrhizobium japonicum (8317 genes). The average gene content is 3,100 genes per genome.
[NB: Bradyrhizobium japonicum is a species of legume - root nodulating, microsymbiotic
nitrogen-fixing bacteria].
- Analysis of Microbial Genome Based on Clusters of Orthologous Genes (COG) or
Clusters of Orthologous Groups of Proteins:
-Cluster of Orthologous Genes (COG) describe a sets of genes that have descended from a single
common ancestor (common evolutionary descent) within a taxonomic range of microorganisms
—i.e.—a collection of homologous genes used in the study evolutionary (phylogenetic)
relationships among microorganisms. Clusters of Orthologous Genes (COG) database is a
popular tool and was created for the following purposes: (i) Microbial genome annotation (i.e.
description or elucidation) and comparative genomics; (ii) Evolutionary classification of
microbial protein families; (iii) Unification of genome annotation in groups of related
organisms; (iv) Identification of missing and/or undetected genes in complete microbial
genomes; (v) Analysis of genomic neighborhoods, thus helping prediction of novel genomic
functions in microbial systems; (vi) Analysis of metabolic pathways and prediction of
alternative forms of enzymes.
- General Features of Bacterial Chromosomes :
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-Not all bacteria have a single circular chromosome. Some bacteria have multiple circular
chromosomes. Many bacteria have linear chromosomes and linear plasmids. Linear
chromosomes and plasmids were not discovered in bacteria until relatively recently. For
instance, Borrelia burgdorferi, the bacterium that causes Lyme disease contains up to 11 copies
of a single linear chromosome. Agrobacterium tumefaciens contains one linear (2.1 Mb)
chromosome and one circular (3 Mbp) chromosome, plus two circular plasmids (450 Kbp + 200
Kbp). Vibrio cholerae, the bacterium that causes cholera, possesses two circular chromosomes.
One of these chromosomes contains the genes involved in metabolism and virulence, while the
other contains the remaining essential genes. Bacillus thuringiensis contains one circular (5.7
Mbp) chromosome and six plasmids (each > 50 Kbp). Escherichia coli, model organism of
choice for prokaryotic research possesses a single circular chromosome packaged within the
cell nucleoid. The term nucleoid means nucleus-like.
- Genome Packaging in Prokaryotes :
-Prokaryotic cells do not contain nucleus (plural: nuclei) or other membrane-bound organelles.
The term “prokaryote” means “before the nucleus”. All or most of the genetic material
(bacterial chromosome) of prokaryotic cells are normally localized (located) within an
irregularly-shaped region known as nucleoid.
-Experimental evidence suggests that the nucleoid is largely composed of about 60% DNA, plus
a small amount of RNA (mainly mRNA) and proteins (mostly transcription factor proteins).
The messenger RNA and the transcription factor proteins are important in regulating activities
of the bacterial genome.
-Since the genomes of prokaryotic organisms are generally circular, proteins helping to maintain
the supercoiled structure of their nucleic acids (i.e. DNA compaction and genome organization)
are known as nucleoid proteins or nucleoid-associated proteins, and are distinct from histones
of eukaryotic nuclei.
- DNA Supercoiling :
-Whereas eukaryotes wrap their DNA around proteins called histones to help package the DNA
into smaller spaces, most prokaryotes do not have histones (with the exception of those species
in the domain Archaea). Thus, one way prokaryotes compress their DNA into smaller spaces is
through supercoiling.
-DNA supercoiling refers to the over- or under-winding of a DNA strand. Supercoiling is
important in a number of biological processes, such as compacting DNA. Additionally, certain
enzymes such as topoisomerases are able to change DNA topology to facilitate functions such
as DNA replication or transcription.
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-Genomes can be negatively supercoiled, meaning that the DNA is twisted in the opposite
direction of the double helix, or positively supercoiled, meaning that the DNA is twisted in the
same direction as the double helix.
-As a general rule, the DNA of most organisms is negatively supercoiled (i.e. most bacterial
genomes are negatively supercoiled during normal growth).
- The Importance of DNA supercoiling :
i. DNA supercoiling is important for DNA packaging within all cells. Because the length of
DNA can be thousands of times that of a cell, packaging this genetic material into the cell or
nucleus is significant to both prokaryotes and eukaryotes. Supercoiling of DNA reduces the
space and allows for much more DNA to be packaged. ii. Supercoiling is also required for
DNA and RNA synthesis—i.e.—supercoiling facilitates unwinding of DNA during replication
for the action of DNA and RNA polymerase enzymes. This include both positive and negative
supercoiling. Moreover, topoisomerases such as DNA gyrase (Type II Topoisomerase) enhance
supercoiling during DNA and RNA synthesis.
- Proteins Involved in Supercoiling :
-Multiple proteins act together to fold and condense prokaryotic DNA:
i. HU protein: is the most abundant protein in the nucleoid and works together with
topoisomerase I enzyme to bind DNA and introduce sharp bends in the chromosome,
generating the tension necessary for negative supercoiling.
ii. Integration host factor (IHF): binds to specific sequences within the genome to
introduce additional bends. The folded DNA is then organized into a variety of
conformations that are supercoiled and condensed—leading to stability of chromosomal
DNA. iii. Histone-like nucleoid-structuring (H-NS) protein: works together with
DNA topoisomerase I and DNA topoisomerase II (DNA gyrase) to help maintain the
supercoiled DNA.
iv. Factor for inversion stimulation (FIS): is abundant during exponential growth and
regulates the expression of more than 231 genes, including DNA topoisomerase I.
Variation in Genomic Constitution:
-It is imperative to note that: “prokaryotic genomes are smaller and less variant than eukaryotic
genomes”.
-Why are prokaryotic (bacterial) genomes smaller and less variant than eukaryotic genomes ?
1. Bacteria have relatively small amounts of junk DNA (i.e. non-coding regions), most of the
bacterial genomes (85-90%) is non-repetitive DNA (coding DNA).
2. A large number of bacterial species have undergone genomic degradation resulting in a
decrease in genome size from their ancestral state;
3. The lifestyles of bacteria play an integral role in their respective genome sizes:
-Facultative and recently evolved pathogenic bacteria exhibit a smaller genome size than free-
living bacteria, yet they have more pseudogenes than any other form of bacteria;
-Obligate bacterial symbionts or pathogens have the smallest genomes and the fewest number of
pseudogenes than any other form of bacteria. The relationship between lifestyles of bacteria and
genome size raises questions as to the mechanisms of bacterial genome evolution.
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A pseudogene is a segment of DNA that structurally resembles a gene but is not capable
of coding for a protein.
4. Bacteria have smaller genomes due to a selective pressure on genome size to ensure faster
replication—smaller bacterial genomes will take less time to replicate. Subsequently,
smaller genomes will be selected preferentially due to enhanced fitness.
5. Bacterial genes are generally packed closer together than in most eukaryotes with relatively
few intergenic spacer regions. This helps reduce the size of bacterial genomes.
6. DNA supercoiling helps prokaryotes compress their DNA into smaller spaces; allowing for
much more DNA to be packaged hence reducing prokaryotic genome sizes.
7. Most prokaryotes contain only one copy of their genes/chromosomes (i.e. they are
haploids).
Key Genomic (chromosomal) Features of E. coli :
-Escherichia coli, a widely used bacterium in molecular biology research has the following
genomic features:
1. The bulk of the DNA (genome) in E. coli cells consists of a single (haploid) double-stranded
closed-circular DNA molecule of 4.7 million base pairs in length (i.e. 4,700 kb in size), with
no free 5′ or 3′ ends.
2. The chromosomal DNA is organized into a condensed ovoid structure known as nucleoid.
3. The genome of E. coli contains approximately 4,300 potential coding sequences, and only
about 1,800 known E. coli proteins; and 122 structural RNA genes.
4. Prokaryotic (bacterial operons e.g. E. coli) contain multiple polycistronic transcription units
(or genes)—where a single structural gene or mRNA encodes for multiple proteins or
polypeptides. In contrast, eukaryotic mRNA is monocistronic, since it codes for a single
protein or polypeptide.
5. E. coli usually undergoes asexual reproduction.
6. Transcription and translation processes in E. coli occurs almost simultaneously in the same
compartment (i.e. in the cytoplasm).
TOPIC: 6
PROKARYOTIC DNA REPLICATION:
Key Points:
-DNA replication is semi-conservative. Each strand in the double helix acts as a template for
synthesis of a new complementary strand.
-New DNA is made by enzymes called DNA polymerases, which require a template and a primer
(starter) and synthesize DNA in the 5' to 3' direction
-During DNA replication, one new strand (the leading strand) is made as a continuous piece. The
other (the lagging strand) is made in small pieces.
-DNA replication requires other enzymes in addition to DNA polymerase, including DNA
primase, DNA helicase, DNA ligase, and topoisomerase etc.
Introduction
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-DNA replication , or the copying of a cell's DNA, is detailed process. There are about 3 billion
base pairs of DNA in human genome, all of which must be accurately copied when any one of
the trillions of cells divides.
-The basic mechanisms of DNA replication are similar across organisms. In this lecture, we'll
focus on DNA replication in a prokaryotic cell (bacterium E. coli), but the mechanisms of
replication are similar in humans and other eukaryotes.
- We’ll start by looking at the proteins and enzymes that carry out replication; seeing how
they work together to ensure accurate and complete replication of DNA.
Basic Information
-DNA replication is semiconservative, meaning that each strand in the DNA double helix acts as
a template for the synthesis of a new, complementary strand.
-This process takes us from one starting molecule to two "daughter" molecules, with each newly
formed double helix containing one new and one old strand.
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-The above figures represent the basic process of DNA replication. The most important and
interesting part is how this process is carried out in a cell.
-Cells need to copy their DNA very quickly, and with very few errors. To do so, the cells use a
variety of enzymes and proteins, which work together to make sure DNA replication is
performed smoothly and accurately
DNA polymerase
-One of the key molecules in DNA replication is the enzyme known as DNA polymerase. DNA
polymerases are responsible for synthesizing DNA: they add nucleotides one by one to the
growing DNA chain, incorporating only those that are complementary to the template.
-Here are some key features of DNA polymerases :
They always need a template;
They can only add nucleotides to the 5' end of a DNA strand;
They can't start making a DNA chain from scratch, but require a pre-existing chain or short
stretch of nucleotides called a primer;
They proofread, or check their work, removing the vast majority of "wrong" nucleotides
that are accidentally added to the chain.
-The addition of nucleotides requires energy. This energy comes from the nucleotides
themselves, which have three phosphates attached to them (much like the energy-carrying
molecule ATP). When the bond between phosphates is broken, the energy released is used to
form a bond between the incoming nucleotide and the growing chain.
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-In prokaryotes such as E. coli, there are three main DNA polymerases involved in DNA
replication: DNA pol I, DNA pol II, and DNA pol III. DNA pol III is the enzyme required for
DNA synthesis; DNA pol I and DNA pol II are primarily required for repair.
Starting DNA Replication
-How do DNA polymerases and other replication factors know where to begin?
-Replication always starts at specific locations on the DNA, which are called origins of
replication and are recognized by their sequence.
-E. coli, like most bacteria and archaea, have single origins of replication on their chromosomes.
The replication origins mostly have A-T base pairs (which are held together by fewer hydrogen
bonds than G-C base pairs), making the DNA strands easier to separate.
-Specialized proteins recognize the origin, bind to this site, and open up the DNA. As the DNA
opens, two Y-shaped structures called replication forks are formed, together making up what's
called a replication bubble. The replication forks will move in opposite directions as replication
proceeds.
-How does replication actually get going at the forks?
- Helicase is the first replication enzyme to load on at the origin of replication. Helicase's job is
to move the replication forks forward by "unwinding" the DNA (breaking the hydrogen bonds
between the nitrogenous base pairs).
- Proteins called single-strand binding proteins coat the separated strands of DNA near the
replication fork, keeping them from coming back together into a double helix.
Primers and Primase
-DNA polymerases can only add nucleotides to the 5' end of an existing DNA strand. How, then,
does DNA polymerase add the first nucleotide at a new replication fork?
-This is achieved by the help of an enzyme known as primase. Primase makes an RNA primer
(short stretch of nucleic acid complementary to the template); that provides a 3' end for DNA
polymerase to work on.
-The primer ‘primes’ DNA synthesis , i.e., gets it started.
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-Once the RNA primer is in place, DNA polymerase "extends" it, adding nucleotides one by one
to make a new DNA strand that's complementary to the template strand.
Leading and Lagging Strands
-In E. coli, the DNA polymerase that participates in most of the synthesis is DNA polymerase
III. There are two molecules of DNA polymerase III at a replication fork; each of them works
on one of the two new DNA strands.
-DNA polymerases can only make DNA in the 5' to 3' direction. A DNA double helix is always
anti-parallel; in other words, one strand runs in the 5' to 3' direction, while the other runs in the
3' to 5' direction. This makes it necessary for the two new strands, which are also antiparallel to
their templates, to be made in slightly different ways.
-One new strand, which runs 5' to 3' towards the replication fork, is the easy one. This strand is
made continuously, because the DNA polymerase is moving in the same direction as the
replication fork. This continuously synthesized strand is called the leading strand.
-The other new strand, which runs 5' to 3' away from the fork, is trickier. This strand is made in
fragments because, as the fork moves forward, the DNA polymerase (which is moving away
from the fork) must come off and reattach on the newly exposed DNA. This tricky strand,
which is made in fragments, is called the lagging strand.
-The small fragments are called Okazaki fragments, named for the Japanese scientist who
discovered them. The leading strand can be extended from one primer alone, whereas the
lagging strand needs a new primer for each of the short Okazaki fragments.
Maintenance and Cleanup during the Replication Process
-Some other proteins and enzymes, in addition to the main ones above, are needed to keep DNA
replication running smoothly. One is a protein called the sliding clamp, which holds DNA
polymerase III molecules in place as they synthesize DNA. The sliding clamp is a ring-shaped
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protein and keeps the DNA polymerase of the lagging strand from floating off when it re-starts
at a new Okazaki fragment.
- Topoisomerase also plays an important maintenance role during DNA replication. This enzyme
prevents the DNA double helix ahead of the replication fork from getting too tightly wound as
the DNA is opened up. It acts by making temporary nicks (notch/groove) in the helix to release
the tension, then sealing the nicks to avoid permanent damage.
-Finally, there is a little cleanup work to do if we want DNA that doesn't contain any RNA or
gaps. The RNA primers are removed and replaced by DNA through the activity of DNA
polymerase I, the other polymerase involved in replication. The nicks (grooves/gaps) that
remain after the primers are replaced get sealed by the enzyme DNA ligase.
Summary of DNA Replication in Prokaryotes ( E. coli )
-The process of DNA replication can be summarized as follows :
1. DNA unwinds at the origin of replication.
2. Helicase opens up the DNA-forming replication forks; these are extended in both directions
(i.e. bidirectionally).
3. Single-stranded binding proteins coat the DNA around the replication fork to prevent
rewinding of the DNA.
4. Topoisomerase binds at the region ahead of the replication fork to prevent supercoiling
(overwinding).
5. Primase synthesizes RNA primers complementary to the DNA strand.
6. DNA polymerase III starts adding nucleotides to the 3′-OH end of the primer.
7. Elongation of both the lagging and the leading strand continues. 8. RNA primers are
removed by exonuclease activity 9. Gaps are filled with DNA by DNA pol I.
10. The gaps between the two DNA fragments are sealed by DNA ligase.
Summary: Prokaryotic DNA Replication: Enzymes and Their Function
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Exonuclease activity removes RNA primer and replaces with
DNA pol I
newly synthesized DNA
DNA pol II Repair function
DNA pol III Main enzyme that adds nucleotides in the 5′-3′ direction
Opens the DNA helix by breaking hydrogen bonds between the
Helicase nitrogenous bases (i.e. responsible for DNA unwinding).
Seals the gaps between the Okazaki fragments to create one
DNA ligase
continuous DNA strand
Primase Synthesizes RNA primers needed to start replication.
Helps to hold the DNA polymerase in place when nucleotides
Sliding Clamp are being added
Works at the region ahead of the replication fork to prevent
Topoisomerase supercoiling.
Single-strand binding proteins Coats or binds to single-stranded DNA to avoid DNA rewinding
(SSB) back.
Gene Expression:
-Gene expression involves two separate but inter-related processes, transcription and
translation.
A. Transcription: Transcription is the process of synthesizing RNA from a DNA template. The
DNA acts as a template for the transcription of RNA by RNA polymerase for subsequent
protein production within the cell. RNA polymerase attaches itself to the beginning of a
gene on DNA and synthesizes mRNA, using one of the strands in DNA as a template. This
process is known as transcription. The bases in mRNA will be complementary to one
strand of DNA since DNA acts as a template for synthesis of mRNA.
B. Translation: Translation is the process of decoding the information carried on the mRNA to
synthesize the specified protein
- Process of translation: The process of translation (in prokaryotes, mainly bacteria)
requires three major components, namely mRNA, ribosomes, and tRNAs in addition to
various accessory proteins.
- Messenger RNA (mRNA) : The mRNA is a temporary copy of genetic information. It
carries the coded information for making specific proteins from DNA to ribosomes, where
proteins are synthesized.
- Ribosomes : Serve as the sites of translation, and their structure facilitates the joining of one
amino acid to another.
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Enzyme/protein Specific Function
- Transfer RNA (tRNA ): The tRNA molecule contains a triplet (codon) at one end and
amino acid at the other end. The ribosome moves along the mRNA until the entire mRNA
molecule has been translated into corresponding sequences of amino acids. Finally, the
sequence of amino acids in the resulting polypeptide chain determines the configuration into
which the polypeptide chain folds itself, which in many cases determines the enzymatic
properties of the completed protein.
TOPIC: 7
BACTERIA (PROKARYOTIC) GENE REGULATION:
Key Points:
-Bacterial genes are often found in operons. Genes in an operon are transcribed as a group and
have a single promoter.
-Each operon contains regulatory DNA sequences, which act as binding sites for regulatory
proteins that promote or inhibit transcription.
- Regulatory proteins often bind to small molecules, which can make the protein active or
inactive by changing its ability to bind DNA.
-Some operons are inducible, meaning that they can be turned on by the presence of a particular
small molecule. Others are repressible, meaning that they are on by default but can be turned
off by a small molecule.
Introduction
- Bacteria may seem simple, however, if even the simplest bacterium has a complex task when
it comes to gene regulation! The bacteria in our body have genomes that contain thousands of
different genes. Most of these genes encode proteins, each with its own role in a process such as
fuel metabolism, maintenance of cell structure, and defense against viruses.
-Some of these proteins are needed routinely, while others are needed only under certain
circumstances. Thus, cells don't express all the genes in their genome all the time. You can
think of the genome as being like a cookbook with many different recipes in it . The cell will
only use the recipes (express the genes) that fit its current needs.
How is Gene Expression in Bacteria Regulated?
-There are various forms of gene regulation, (i.e. mechanisms for controlling which genes get
expressed and at what levels).
- Note: bacterial gene regulation occurs at the four levels as indicated below.
-Bacteria have specific regulatory molecules that control gene regulation (i.e. regulatory
molecules that determine whether a particular gene will be transcribed into mRNA).
-Normally, regulatory molecules act by binding to DNA near the gene hence facilitate (help) or
inhibit (block) the transcription of RNA polymerase enzyme .
- Let's take a closer look at how genes are regulated in bacteria; which is basically similar to
archaeal gene regulation.
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In Bacteria, Genes are often found in Operons
-As already mentioned, related genes in bacteria are often found in a cluster on the chromosome;
where they are transcribed from one promoter (RNA polymerase binding site) as a single unit.
[Such a cluster of genes under control of a single promoter is known as an operon].
-Operons are common in prokaryotes (bacteria/archaea), but they are rare in eukaryotes such as
humans.
-In general, operons contain genes that function in the same process.
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-An example of operon is the lac operon; which contains genes that encode proteins involved in
uptake and metabolism of sugar lactose.
-NB: operons allow the cell to efficiently express sets of genes whose products are needed at the
same time.
Structure of an Operon
-Operons are made up of coding sequences of genes and regulatory DNA sequences whose
function is to control transcription of the operon.
-These sequences are binding sites for regulatory proteins, which control how much the operon
is transcribed. The promoter, or (site where RNA polymerase binds), is one example of a
regulatory DNA sequence.
-In addition to the promoter, most operons have other regulatory DNA sequences. These
sequences are binding sites for regulatory proteins that turn expression of the operon “up/on”
or
“down/off.”
Some regulatory proteins are repressors that bind to pieces of DNA known as operators.
When bound to its operator, a repressor reduces (suppresses) transcription (e.g., by blocking
RNA polymerase from moving forward on the DNA).
Some regulatory proteins are activators. When an activator is bound to its DNA binding
site, it increases transcription of the operon (e.g., by helping RNA polymerase bind to the
promoter).
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-Where do the regulatory proteins come from ? Like any other protein produced in an organism,
they are encoded by genes in the bacterium's genome. The genes that encode regulatory proteins
are sometimes called regulatory genes.
-Many regulatory proteins can themselves be turned "on" or "off" by specific small molecules .
The small molecule binds to the protein, changing its shape and altering its ability to bind DNA.
For instance, an activator may only become active (able to bind DNA) when it's attached to a
certain small molecule.
Operons May Be Inducible or Repressible
-Some operons are usually “off”, but can be turned “on” by a small molecule. The molecule is
known as an inducer, and the operon is said to be inducible.
For example, the lac operon is an inducible operon that encodes enzymes for metabolism
of the sugar lactose. It turns on only when the sugar lactose is present (and other, preferred
sugars are absent). The inducer in this case is allolactose, a modified form of lactose.
-Other operons are usually “on”, but can be turned “off” by a small molecule. The molecule is
known as a corepressor, and the operon is said to be repressible.
For example, the trp operon is a repressible operon that encodes enzymes for synthesis of
the amino acid tryptophan. This operon is expressed by default, but can be repressed when
high levels of the amino acid tryptophan are present. The corepressor in this case is
tryptophan.
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- These examples illustrate an important point: that gene regulation allows bacteria to respond
to changes in their environment by altering gene expression (and thus, changing the set of
proteins present in the cell).
Some Genes and Operons are Expressed All the Time
-Many genes play specialized roles and are expressed only under certain conditions, as described
above. However, there are also genes whose products are constantly needed by the cell to
maintain essential functions (e.g. DNA replication, repair, and expression). The genes are
referred to as housekeeping genes.
-These housekeeping genes are constantly expressed (i.e. "constitutively active") under normal
growth conditions. [Housekeeping genes have promoters and other regulatory DNA sequences
that ensure constant expression].
- NB: in summary, the general structure of an operon includes:
Promoter: The promoter is located at the upstream of the structural genes and is the site
recognized by the RNA Polymerase and initiates transcription.
Operator: The segment of DNA located in between the structural genes and the promoter.
The site that is recognized by the repressor and hence acts as the physical barrier for the
RNA polymerase thus controlling the transcription.
Structural genes: Genes that are involved in making of proteins.
TOPIC: 8
EXTRACHROMOSOMAL GENETIC ELEMENTS (PLASMIDS):
-The term plasmid was first introduced by the American molecular biologist Joshua Lederberg in
1952. A plasmid is a DNA molecule that is separate from, and can replicate independently (of the
chromosomal DNA. In other words, a plasmid is a small DNA molecule within a cell that is
physically separated from chromosomal DNA and can replicate autonomously or independently.
-Plasmids are double-stranded and, in many cases, they occur as closed circular molecules;
however, they may occur as linear molecules as seen in the bacterial species Borrelia burgdorferi.
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-They are regarded as extrachromosomal genetic elements (or DNA) containing extrachromosomal
genome.
-Plasmids usually occur naturally in bacteria, but are sometimes found in archaea and eukaryotic
organisms.
-A bacterial cell can have no plasmids at all or may possess one or many/multiple copies of one
or more plasmids.
-Plasmids are inherited from parent bacterial cell to daughter cell and have capability to
selfreplication in the cytoplasm of bacterial cell. The circular molecule of DNA can be broken to
yield a linear molecule which passes from one bacterial cell to another.
-There are many kinds of plasmids which differ from one another in size and in composition of
genes. They vary in size from 1 Kbp to more than 1,000 Kbp.
-Plasmids are transmitted from one bacterium to another (even of another species) mostly through
conjugation. This host-to-host transfer of genetic material is one mechanism of horizontal
gene transfer, and thus, plasmids are considered part of the mobilome (i.e. transposable
elements).
-Unlike viruses, which encase their genetic material in a protective protein coat called a capsid,
plasmids are ‘naked’ DNA and do not encode genes necessary to encase the genetic material for
transfer to a new host. However, some classes of plasmids encode the conjugative ‘sex’ pilus
necessary for their own transfer.
-Plasmids can exist as episomes (i.e. get integrated into the host bacterium’s chromosomal DNA)
or occur as non-integrated plasmids. Figures 1 & 2 below outline ‘step by step’ mechanisms of
cloning genes using plasmids: comparing activity of non-integrating plasmids with integrated
plasmids (episomes) during cell division.
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-The major components of a plasmid comprise of an origin site (origin of replication) or a specific
portion of their genome that serves as start signal for self-replication. Under natural conditions,
each plasmid replicates to produce 20-30 copies per cell. This number can be artificially increased.
In presence of certain antibiotics, the number can be increased to about 1000 copies.
-Another valuable feature of plasmids is the presence of specific restriction sites where the
enzyme restriction endonuclease makes a cut so that a foreign DNA segment may be jointed to the
plasmid. This property helps plasmids to act as trusted cloning vehicles during gene transfer in
genetic engineering.
-Plasmids are considered replicons—units of DNA or RNA capable of replicating autonomously
within a suitable host.
-In nature, plasmids often carry genes that benefit the survival of the host organism (bacteria). For
instance, plasmids may carry genes that provide resistance to naturally occurring antibiotics in
a competitive environmental niche, or the proteins produced may act as toxins under similar
circumstances. Plasmids can also provide bacteria with the ability to fix elemental nitrogen or to
utilize/degrade recalcitrant organic compounds that would be advantageous when nutrients are
scarce.
-While the chromosomes are big and contain all the essential genetic information for living under
normal conditions, plasmids usually are very small and contain only additional genes that may be
useful in certain situations or conditions.
-Artificial plasmids are widely used as vectors in molecular cloning, serving to drive the
replication of recombinant DNA sequences within host organisms. In molecular cloning, a vector
is a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell,
where it can be replicated and/or expressed.
- Key Terms :
-Plasmid: A circle of double-stranded DNA that is separate from the chromosomes, which is found
in bacteria, archaea and eukaryotes.
-Mobilome: The entirety of the mobile (transposable) elements of a genome. - Replicon:
•A region of DNA or RNA, that replicates from a single origin of replication OR; A
unit of DNA or RNA capable of replicating autonomously within a suitable host.
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-Episome:
•A segment of extrachromosomal DNA (or plasmid) that can become incorporated into
the chromosome of a host cell OR;
•A plasmid DNA that can be inserted (or integrated) into the bacterial chromosomal DNA
and can replicate with it.
•NB: The term episome can be applied to both plasmids and viral genomes.
Structure/Components of Plasmids:
-Every plasmid has certain essential elements or important parts. These include:
- Origin of replication (Ori): This refers to a specific location in the strand where the replication
process begins. In plasmids, this region is largely made up of A-T base pairs as it is easier to
separate (or split off) the strands during replication. Origin of replication denotes DNA sequence
which allows bacteria to make more copies of the plasmid as they grow and divide. Origin of
replication is also known as replicon.
- Selectable marker site: This region consists of antibiotic resistance genes which are useful in the
identification and selection of bacteria that contain the desired plasmids or plasmids of interest.
- Promoter region: This is where transcriptional machinery proteins (such as RNA polymerase and
transcription factors e.g. activators, repressors, enhancers, silencers etc.) are loaded or bind to
initiate transcription of the target gene.
- Primer binding site: this is a short sequence of single-stranded DNA which is used as an
initiation point for DNA amplification and DNA sequencing. A primer binding site also refers to
the region of a nucleotide sequence where an RNA or DNA single-stranded primer binds to start
replication.
- Multiple cloning sites: Also known as polylinkers, multiple cloning sites (MCS) are short
segments of DNA which contain many (up to ~20) restriction sites (or restriction recognition
sites) for a number of restriction enzymes. Polylinkers facilitate DNA insertion via restriction
enzyme digestion or ligation—every restriction enzyme has the capacity to cut/cleave the plasmid
at MCS and allow DNA insertion.
- Antibiotic resistance gene: It is a specific gene that allows bacteria with the plasmid to grow in
the presence of an antibiotic specific to the gene—hence confer drug resistance property to
bacteria.
- Insert: Gene, promoter or other DNA fragment cloned into the MCS for further study.
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Properties/Characteristics of Bacterial Plasmids:
- Double stranded circular DNA:
-Plasmids are double-stranded circular and supercoiled DNA. Exceptions are the linear plasmids in
bacterial species Streptomyces and Borrelia.
- Small size:
-They have small size, which makes them easy to isolate and manipulate.
- Independent origin of replication:
-Plasmids contain genes or signals for independent replication (self-replication) within cells.
- Curing:
-Plasmids can be eliminated from bacterial cells by a process known as curing. Curing may take
place spontaneously or it may be induced by various treatments, which inhibit plasmid replication
but do not affect bacterial chromosome replication and cell reproduction.
- Incompatibility:
-This refers to the ability of two different plasmids to co-exist in the same bacterial cell (i.e. two
members of the same group of plasmid cannot co-exist in the same cell).
-Plasmids are incompatible if they have the same reproduction strategy in the cell; this allows the
plasmids to inhabit a certain territory within the cell without other plasmids interfering.
- Copy number:
-The copy number refers to the number of copies of plasmid present in the bacterial cell. Usually,
small plasmids are present in high numbers and large plasmids are present in few numbers.
-Multiple copy number—makes them to be present in the cell in several copies so that
amplification of the plasmid DNA becomes easy.
- Transferability:
-Some plasmids are self-transferable.
- Recombination:
-Plasmids occur as episomes which can be integrated with host chromosome.
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-
Mobilization:
-By the process of integration, the self-transferable plasmid can mobilize the chromosomal gene
or other plasmids—to carry only a subset of the genes required for transfer.
- Selectable marker: presence of selectable markers such as antibiotic resistance genes—make
detection and selection of plasmid-containing clones easier.
Classification of Plasmids/Types of Plasmids:
1): Based on their functions , plasmids can be classified into five types :
-Resistance (R) plasmids
-Fertility (F) plasmids
-Bacteriocinogen or Col plasmids
-Degradative plasmids
-Virulence plasmids
a): Resistance (R) plasmids (R-factors):
-R-plasmids are circular with double-stranded plasmid.
-They contain genes that provide (or confer) resistance against antibiotics (antibacterial agents)
or poisons. They are also known as R-factors—i.e.—resistance factors.
-These plasmids have been found in enteric bacteria (e.g. E. coli and Shigella dysenteriae) and
thus provide a great threat to the medical science.
-R-plasmids occur in two sizes :
-Large R-plasmids (molecular weights ranging between 30×106 Daltons).
-Small R-plasmids (molecular weights of about 5 to 6×106 Daltons).
-Large R-plasmids are self-transmissible by conjugation to other bacteria—i.e.—they are
conjugative plasmids just like F-plasmids; possessing extra genes (DNA) that code for
conjugation process.
-Small R-plasmids contain only resistance genes—also known as ‘r’ genes. They are
nontransmissible and not conjugative.
-Large R-plasmids e.g. R100 plasmids of Shigella and E. coli have multiple drug resistance
genes for sulfonamides, streptomycin, fusidic acid, chloramphenicol, and tetracycline etc.
-R-plasmids consists of two components :
-Resistance transfer factor (RTF): carries genes that govern the process of intercellular or
conjugal transfer—i.e.—contains ‘transfer genes’ responsible for the transfer functions of
Rplasmids.
-Resistant determinant (r-determinant): carries resistant genes for each of the several drugs—
i.e.—codes for resistance against various drugs.
-The drug resistance property of bacteria is not transferrable in the case when RTF dissociates
or get separated from the r-determinant.
-For the spread of the multiple drug resistance in the bacteria, R-factors (R-plasmids) play a
vital role.
-R-plasmids enable bacteria to destroy antibiotics and to modify the membrane transport
system, thus inactivating antibacterial effects of drugs. For instance, plasmids carrying the gene
for the enzyme β-lactamase confer resistance to β–lactam antibiotics such as penicillin and
amoxicillin.
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-
Besides drug resistance, R-plasmids may also make bacterial hosts resistant to the toxic effects
of heavy metals such as Nickel, Cobalt, Mercury, Arsenic and Cadmium etc.
b): F-plasmids (Fertility Factor):
-Fertility plasmids (F-plasmids) are also known as fertility factors (F-factors) or sex factors.
-They contain transfer factors or transfer ( tra ) genes that allow genes to be transferred from one
bacteria to another through conjugation. Thus, F-plasmids are conjugative plasmids.
-In other words, F-factor is a plasmid that codes for sex pili and its transfer to other bacterial
cells.
-Strains of bacteria possessing F-plasmids are regarded as F positive (F+) and function as donors
(male). Such bacteria have sex pili on their surfaces.
-Strains of bacteria lacking the F-plasmid are designated F negative (F-) and function as
recipients (female).
-An F- bacterium will become F+ bacterium when it conjugates or receives the fertility factor
from another F+ bacterium. This will lead to the formation of two F+ bacterial cells.
-The conjugative property of F-plasmids is determined by a cluster of at least 25 transfer (tra)
genes which perform the following functions : - Determine expression and synthesis of sex-
pili;
-Determine synthesis and transfer of DNA during mating—initiating self-transfer or gene
transfer;
-Enhance extra-chromosomal existence of F-plasmids;
-Control plasmid replication;
-Interference with the ability of F+ bacteria (donor) to serve as recipients (F-).
-Other key / salient features or characteristics of F-plasmids include :
-F-plasmids are episomes—i.e.—plasmids that can be inserted (become incorporated) into
chromosomal DNA of the host cell.
-F-plasmids have two origins of replication (i.e. oriV and oriS) associated with bidirectional and
unidirectional replication, respectively.
-When outside the chromosome, F plasmids replicate as do bacterial chromosomes, i.e.,
replication begins at an origin of replication (in this case, oriV) and proceeds in both directions
around the closed loop.
-When integrated into the chromosome , F plasmid replicates in a unidirectional manner with
replication initiated at oriS.
-F plasmid also includes an origin of transfer (oriT) located midway along its length when the
plasmid is integrated in the host chromosome (NB: oriT is a short sequence necessary for the
transfer of plasmid DNA from a bacterial donor to recipient during bacterial conjugation).
c): Bacteriocinogens or Col-plasmid (Colicin plasmids):
-Col-plasmids are also known as bacteriocinogenic plasmids or bacteriocinogens; because
they produce bacteriocins. Bacteriocins are proteins or peptide toxins produced by bacteria to
kill or inhibit the growth of similar or closely related bacterial species or different strains of the
same species.
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-
-Different strains of coliform bacteria (mostly E. coli) possess Col-plasmids—containing genes
that control synthesis of a class of proteins known as colicins. Colicins are types of bacteriocins
that are capable of inhibiting growth of related bacteria which lack Col-plasmids.
Colicin production is determined by plasmids known as colicinogenic (Col) factors—which
resemble the F-factors in promoting conjugation, leading to self-transfer and, at times, transfer
of chromosomal segments from one bacterial cell to another.
-Other than colicins produced by E. coli, many bacteria produce bacteriocins which are able to
kill other related or even unrelated bacteria. Such bacteriocins include:
- Pyocin produced by Pseudomonas pyocyanea is also known as Pseudomonas aeruginosa;
- Megacin produced by Bacillus megaterium;
- Marscesins produced by Serratia marcescens;
- Diphthericin produced by Corynebacterium diphtheriae; - Nisin produced by
Lactobacillus lactis.
d): Degradative plasmids:
-Degradative plasmids are also known as metabolic/catabolic or dissimilation plasmids.
-They help the host bacteria to digest or degrade recalcitrant (or unusual) organic compounds
that are not commonly found in nature, such as camphor, xylene, toluene, and salicylic acid and
complex hydrocarbons of crude petroleum etc. These plasmids contain genes coding for
enzymes that catabolize, decompose or break-down complex or specific organic molecules.
-With the help of these enzymes, the bacteria can utilize these compounds as source of carbon
and energy. For example, in species of Pseudomonas putida, both chromosomal and plasmid
genes produce enzymes for break-down of complex compounds.
-As a result, bacteria possessing such degradative plasmids stand a much better chance of
survival under conditions where only such unusual compounds are available. Normal bacteria
without such plasmid-coded enzymes would perish under similar conditions.
-The capability of organisms carrying degradative plasmids to metabolize unusual diverse
complex compounds suggests the possibility of employing them as means of bioremediation
of the polluted environment. For instance, a synthetic strain of Pseudomonas species has been
developed with the prospect of practical utilization in removing oil-spills in the oceans, caused
by leakage of crude petroleum from tankers. Oil-spills are a great danger to marine life, both
plants and animals.
e): Virulence plasmids:
-Virulence plasmids confer pathogenicity on the host bacteria—turning the bacteria into
pathogens. They make the bacteria more pathogenic, as the bacteria are able to resist host
defense or to produce toxins and other virulence factors—thus causing disease. The pathogenic
bacteria can easily be spread and replicated among affected individuals.
- Examples:
- Tumor-inducing plasmids (Ti-plasmids) of Agrobacterium tumefaciens (plant pathogenic
bacteria) induce crown gall disease in many dicotyledonous plant species. Crown gall disease
can be identified by the appearance of tumors of various size and shape at lower stem and main
roots of the plants. The tumor cells use plant metabolites to form nutrients for bacterial growth.
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-
-The bacteria E. coli have several virulence plasmids that code for enterotoxins which induce
severe diarrhea and vomiting.
-Salmonella enterica is another bacterium that contains virulence plasmids—causing typhoid
fever or enteric fever.
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2): Based on their roles in conjugation , plasmids are of two types :
-Conjugative plasmids
-Non-conjugative plasmids
a): Conjugative plasmids:
-Conjugative plasmids are also known as self-transmissible plasmids.
-These plasmids carry transfer (tra) genes which perform the complex process of conjugation—
the transfer of plasmids to another bacterium. Thus, they are F-plasmids.
-They contain genes that direct the synthesis of sex pili.
-They possess both resistance transfer factor (RTF) and resistant determinant (r-determinant).
b): Non-conjugative plasmids:
-Non-conjugative plasmids are also known as non-self-transmissible plasmids.
-These plasmids lack tra genes thus incapable of initiating conjugation; hence they can be
transferred only with the assistance of conjugative plasmids.
-They lack RTF, but possess only r determinants. This occurs when RTF dissociates or separates
from r-determinant. In such cases, the host cell remains drug resistant but the resistance is not
transferable.
3): Plasmids can also be classified by incompatibility testing :
-This method relies on the fact that closely related plasmids are unable to coexist stably in the
same bacterial cell, while unrelated plasmids can. Plasmids which are sufficiently closely
related normally interfere with each other’s replication and are said to be incompatible and to
belong to the same incompatibility group. In contrast, unrelated plasmids can coexist stably and
are therefore said to belong to different incompatibility groups.
-All members of the same plasmid incompatibility group produce the same type of pilus for
conjugation or share the same replication mechanism.
Other types of plasmids include:
-Recombinant plasmids - Plasmids that have been altered in the laboratory and introduced into
the bacteria for the purposes of studies.
-Suicide plasmids – Plasmids that fail to replicate when transferred from one cell to another
Functions/Significance/Applications of Plasmids:
-The main function of the plasmid is the spread of antibiotic-resistant genes. These resistant
genes are carried within the plasmid and are transferred from one cell to another.
-Plasmids are used in genetic engineering to amplify, or produce many copies of certain genes.
-Plasmids are used in recombinant DNA technology—they can be used as vectors to insert a
specific gene into other organisms due to their capacity to incorporate a gene and replicate
inside the cell.
-Plasmids are used in drug delivery—to deliver or insert desired drug e.g. human insulin into the
body— another useful property of plasmids as vectors.
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-Plasmids are used in gene therapy—to insert therapeutic genes into the human body. This helps
in fighting against diseases—by targeting defective cells and triggering therapeutic genes in
them.
-Plasmids can be used to insert human insulin into the body.
-They can also be used to insert human growth hormone in mammalian cells of animals.
-Degradative plasmids carry the genes involved in metabolic/catabolic activities. They aid in the
digestion of pollutants from the environment—degradation and digestion of the dead organic
matter (dead plants and animals).
-Plasmids can be used in cost-effective bulk production of antibacterial proteins (bacteriocins or
antibiotics).
-Virulence plasmids can carry genes that increase the pathogenicity of the bacteria.
-Plasmids code for resistance to UV light in bacteria (i.e. DNA repair enzymes are coded in the
plasmid).
-Plasmids code for the production of toxins (such as enterotoxins by E. coli, Vibrio cholerae,
exfoliative toxin by Staphylococcus aureus and neurotoxin of Clostridium tetani).
-Plasmids code for colonization factors that are necessary for bacterial attachment to surfaces.
For example, plasmids of Yersinia enterocolitica, Shigella flexneri, enteroinvasive Escherichia
coli produce colonization factors to help the above bacteria attach to gastrointestinal wall.
-R-plasmids may also make bacterial hosts resistant to the toxic effects of heavy metals such as
Nickel, Cobalt, Mercury, Arsenic and Cadmium etc.
-Plasmids are used to create transgenic organisms by introducing beneficial genes into host cells.
For example, the Ti-plasmid is used in plant pathology to develop resistance in plants against
diseases such crown gall tumors. The plasmid is rendered avirulent by curing it, prior to its use
as a vector.
-Plasmids are also used to study the role of various gene and gene products in a biological
system. This is achieved by silencing or overexpressing a particular gene and observing the
effects, if any (gene knockout).
TOPIC: 9
TRANSPOSABLE GENETIC ELEMENTS:
-It is a general assumption that genes do not move from one position to the other on the
chromosomes. The genetic mapping is also based on the same assumption. Majority of the
genes obey this assumption and most of the genes occupy fixed positions on the chromosomes.
Thus, overall structure of the genetic map is practically stable.
-However, beginning in 1940s, scientists have found that certain DNA sequences can actually
change positions.
-Some genes move from place to place inserting themselves into a variety of locations, causing
mutations in genes and changing the ways other genes are expressed. These wanderers are
called transposable elements (TEs).
- Transposable genetic elements or transposons or jumping genes are segments of DNA that
have capacity to move from one location in the genome to another, or between genomes in the
same cell.
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-In other words, transposable genetic elements (transposons) are DNA sequences that can change
their position within a genome, sometimes creating or reversing mutations and altering the
cell’s genetic identity and genome size.
-These elements can insert randomly, move from one chromosome site to another, from plasmids
to the chromosomes, and vice versa, and can be moved from one bacterium to another by
conjugation, transformation, or transduction. This mode of genetic transfer or movement is
known as transposition.
-Transposable elements……can also been defined as those elements which cannot replicate
outside the host genome, but which control their own mobility within it.
-These elements are also known by various names as mobile sequences, mobile elements,
genomic parasites, selfish DNA and/or selfish genes.
-Insertion and transposition of these elements may cause rearrangement of genetic material—
leading to deletions, inversions, chromosomal fusions, and/or duplication of the same genetic
material.
-Transposable elements (found in both prokaryotes and eukaryotes) make up a significant
fraction of the genome and are responsible for much of the mass of DNA in eukaryotic cells—
contributing to junk DNA in eukaryotic organisms.
Structure of transposons: what do they carry?
-Transposable elements usually are flanked by repeated base sequences (i.e. inverted repeats
sequences that function as recognition sites for transposons).
-They often carry transposase genes that gives the transposition ability. These genes occur in the
center of transposons.
-They often carry genes that confer antibiotic resistance or some other benefits to the host e.g.
virulence and symbiotic factors or catabolism of xenobiotics.
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Discovery of Transposable Genetic Elements:
-In 1940s - 1950s, American geneticist Barbara McClintock discovered transposable elements
(transposons) in maize (corn) during her studies on maize genetics, for which she was awarded
the Nobel prize for Medicine/Physiology in 1983.
-In her experiments, McClintock observed pigmentation or colour changes in maize—and
concluded that “genes controlling maize colour kept moving from one locus to another
producing different colour patterns”. She called these genes ‘controlling elements’.
-Since transposons can insert within genes or regulatory sequence of a gene, having little
selectivity in their choice of insertion sites, this results in the complete disruption of gene
function or alteration in the expression of a gene. These disruptions led to the discovery of
transposable elements by Barbara McClintock.
Properties/Characteristics of Transposable Elements:
-Some salient features of transposable elements (TEs) include:
1. TEs can modify gene structure hence causing gene expression.
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2. They are mobile genetic materials that many times carry antimicrobial resistance genes.
3. TEs can insert randomly and move from chromosomes to plasmids and vice versa. By
transductions, conjugation, and transformation the TEs can be moved from one bacterium to
another.
4. Transposable elements are DNA sequences that code for enzymes which result in
selfduplication and insertion into a new DNA site.
5. Transposons are involved in transposition events (which include both recombination and
replication), which usually generates two copies of the original transposable elements. One
copy is retained at the parent site while another copy reaches the host chromosome.
6. The integrity of the target genes of TEs is invariably (or further) disrupted by the presence
of these elements.
7. Since TEs carry the genes which are responsible for initiation of RNA synthesis, they could
activate some previously dormant genes.
8. A transposon cannot replicate without the host chromosome as phages or plasmids since it
lacks the site for the origins of replication.
9. TEs contribute to the repetitive sequences in the genome of the organisms. Human genome
contains 50% repetitive sequences—constituting junk DNA.
10. A transposable element can insert at any position in the plasmid or host chromosome since
no homology exists between a transposable element and its target site of insertion.
Transposable elements rarely insert at base-specific target sites.
11. TEs can create novel genes since they cause mobilization, reshuffling, and rearrangements.
12. In a few rare cases, TEs can cause disease due to mutations or genetic polymorphisms.
Mechanisms of Transposition:
-Transposition is a type of genetic recombination in which certain genetic elements move from
one site on DNA to another. This occurs through recombination between the DNA sequences at
the ends of the transposons and a sequence in the host DNA with little sequence selectivity.
-There are three different mechanisms of transposition (i.e. replicative, conservative and
retrotransposition). Most prokaryotes and eukaryotes employ one of two: either replicative or
conservative mechanism of transposition whereas retro-transposition is only employed by
eukaryotes.
1. Replicative transposition or copy paste transposition :
-Replicative transposons are those transposons which at first replicates or make copy of itself
and then insert one copy of it into a new position or location within a chromosome or a
plasmid. Therefore, during replicative transposition, the transposable elements is replicated and
one copy is inserted into new position or site (on the chromosome or on a plasmid) while one
copy remains at original position (i.e. original copy is retained).
-It is also known as copy and paste transposition.
-This transposition is catalyzed by an enzyme known as transposase which is encoded by the
transposable element itself.
-Replicative transposition is present only in prokaryotic organisms.
2. Non-replicative (conservative) transposition or cut and paste transposition:
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-The conservative transposons are those which change position by excision from one position
and then insertion into another position within a chromosome. Therefore, during conservative
transposition, the transposable element is physically cut from its original position and pasted
(or inserted) into another position in a chromosome.
-It is also known as cut and paste transposition.
-This type of transposition is also catalyzed by an enzyme known as transposase which is coded
by the transposable element itself.
-Conservative transposition is present in both prokaryotic and eukaryotic organisms.
3. Retrotransposons:
- The retro-transposons are those transposons, whose RNA is reversed transcribed by
reverse transcriptase enzyme into DNA and the synthesized DNA is inserted into new
position in a chromosome. In other words, a retrotransposon produces RNA molecules
that are reverse-transcribed into DNA molecules; these DNA molecules are
subsequently inserted into new genomic positions.
- The mechanism of such transposition is known as retrotransposition.
- Some of these retrotransposons are related to retroviruses (e.g. HIV) and utilize their
reverse transcriptase enzyme for transposition, such transposable elements are known as
retroposons.
- Retrotransposons are only present in eukaryotic organisms.
Roles of Transposable Elements:
- They have roles such as:
- In shaping the structure of chromosomes.
- In modulating the expression of genes.
- In converting RNA molecules to DNA.
- In changing positions of DNA sequence in genome.
- In carrying gene for enzyme that catalyzes transposition.
- In carrying antibiotic resistance genes.
The Biological Relevance of Transposons:
1. Transposons are present in the genomes of all lifeforms.
2. Almost half of the human genome is derived from transposable elements.
3. Transposon-related sequences can make up huge fractions of the genome of an organism
(50% of human and maize genome).
4. The transposon content in different genomes is highly variable.
Application of Transposable Elements:
1. Transposable elements can be used as a genetic tool for the analysis of gene expression and
protein functioning.
2. Transposons can be used as molecular biology tools to facilitate cloning of genes, identify
regulatory elements, and produce transgenic organisms.
3. They are used in genetic engineering to insert or remove specific genetic sequences, and also
to cause frameshift mutation.
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4. Transposable elements are also a widely used tool for mutagenesis (formation of mutations)
of most experimentally tractable (amenable) organisms.
5. They are used for the reconstruction of phylogenies (genetic relationships) by the means of
presence/absence analyses. Closely related organisms have similar DNA sequences of
transposable elements.
6. They can act as biological mutagens (i.e. causing genetic mutation) in model organisms such
as Arabidopsis thaliana and Escherichia coli.
7. Transposon systems are being studied for use in human gene therapy—to insert specific
sequences of DNA with therapeutic value into genomes of vertebrate animals and human.
8. Many bacterial transposons carry genes for generic antibiotic resistance. These genes
typically produce enzymes that cleave and render antibiotics non-functional.
Negative Effects of Transposable Elements:
-A transposable gene, when inserted into a functional gene, might disable the gene.
-Multiple copies of the same sequence hinder the chromosome pairing during cell division
resulting in chromosome duplication.
-Expression of harmful disease-causing proteins in the transposons affects the normal cellular
function.
TOPIC: 10
MECHANISMS OF GENETIC EXCHANGE:
-There are three types/mechanisms of genetic exchange (gene transfer) or horizontal gene
transfer occurring naturally in association with prokaryotic organisms; these are
transformation, conjugation and transduction.
1): Bacterial Transformation:
-Transformation, sometimes called DNA mediated transformation, is the transfer of DNA from a
dead donor cell to a live recipient cell. Since the donor cells do not have to be present,
transformation can also be considered as a mechanism allowing bacteria to pick up relatively
small segment of foreign or “naked” DNA from the surrounding/extracellular environment.
Naked DNA is non-viral DNA, i.e., DNA not contained within a protein coat. The cells that
have the ability to uptake DNA are known as competent cells. Because of the newly introduced
genes
(DNA), the bacterial cell is phenotypically altered, or, “transformed.”
-Transformation was first observed to occur in Streptococcus pneumoniae by Frederick Griffith
(1928) while he was attempting to develop a vaccine against pneumonia. Griffith recognized
that capsule formation was a factor influencing the virulence (disease-causing ability) of
Streptococcus pneumoniae bacteria. Cells capable of capsule formation are much more likely to
cause serious disease and death than are non-capsule-forming organisms because capsules
render bacteria resistant to phagocytosis by human white blood cells. During his experiments,
Griffith injected mice with bacterial preparations and obtained the following unexpected
results.
1. When live, capsule-forming Streptococcus cells were injected into mice, the mice developed
pneumonia and died.
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2. When live, non-capsule-forming Streptococcus cells were injected into mice, the mice did
not become sick and survived nicely (or until their next injection).
3. When dead (heat-killed), capsule-forming Streptococcus cells were injected into mice, the
mice did not develop pneumonia, and survived nicely.
4. When a combination of live, non-capsule-forming Streptococcus and dead (heat-killed),
capsule-forming Streptococcus cells were injected into mice, the mice developed pneumonia
and died. Since neither of these two preparations alone caused disease, this was an
unexpected finding.
-From the experiments, Griffith was able to determine that live, capsule-forming Streptococcus
pneumoniae were responsible for killing the mice injected with the cell combination described
in step #4 above. The non-virulent organisms initially injected had been transformed into
virulent pathogens. From this experiment, he concluded correctly that bacteria in the genus
Streptococcus were capable of transferring genes from dead donor cells to living recipient cells.
- The Steps Involved in Bacterial Transformation Include :
1. A donor bacterium dies and is degraded.
2. A fragment of DNA (usually about 20 genes long) from the dead donor bacterium binds to
DNA binding proteins on the cell wall of a competent, living recipient bacterium.
3. Nuclease enzymes then cut the bound DNA into fragments.
4. One strand is destroyed and the other penetrates the recipient bacterium.
5. Enzymes (e.g. recA, recB, recC or recD proteins) promote genetic exchange (recombination)
between a fragment of the donor’s DNA and the recipient’s DNA.
- Bacterial Competence :
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-Not all bacteria are capable of taking up DNA from the surrounding environment. Such bacteria
are made artificially competent. This is achieved by using chemicals and electrical pulses.
Chemicals - the cells are chilled and made permeable in the presence of calcium
phosphate or calcium chloride. The cells are then incubated with the DNA and provided
with a heat shock treatment (at 42°C for 60-120 seconds) that causes the DNA to enter
the cells.
Electroporation - the bacterial cells are subjected to electrical pulses to make them
permeable and cause the DNA to enter into the cells.
- Applications Bacterial Transformation :
-The phenomenon of transformation has been widely used in molecular biology. As they are
easily grown in large numbers, transformed bacteria may be used as host cells for the following:
To make multiple copies of DNA, called DNA cloning.
To make large amounts of proteins and enzymes (e.g. human insulin for treating people
with Type I diabetes).
To genetically modify a bacterium or other cell.
In the generation of complementary DNA (cDNA) libraries - cDNA is often used to
clone eukaryotic genes in prokaryotes. When scientists want to express a specific
protein in a cell that does not normally express that protein (i.e., heterologous
expression), they will transfer the cDNA that codes for the protein to the recipient cell.
In DNA linkage studies – to locate/study the closeness of genes or other DNA sequences
to one another on the same chromosome.
Factors Affecting Transformation:
-DNA size and state: sensitive to nucleases (at least 5 × 105 Daltons).
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-Competence of the recipient: the ability to take up DNA from the environment is known as
competence. Only DNA from closely related bacteria (competent cells) would be successfully
transformed.
2): Bacterial Conjugation:
-Conjugation involves the transfer of genetic material through a specific “sex pilus” between
plasmids (i.e. a process of transfer of genetic material from one bacterium to another by cell-
tocell contact). The sex pilus and conjugation machinery proteins are based on a specialized
plasmid known as the F plasmid or F factor (F = fertility or sex factor). This method was
proposed by Joshua Lederberg and Edward Lawrie Tatum in 1946. They discovered that the
F-factor can move between E. coli cells and proposed the concept of conjugation.
-Conjugation can only occur between donor cells (those carrying F plasmids and producing sex
pili) and recipient cells (those lacking F plasmids and not capable of forming pili). Bacteria
that possess F plasmids are regarded as F+ (male) and act as donors; while those lacking F
plasmid are F- (female) and act as recipient.
Mechanisms of Bacterial Conjugation:
-Bacterial conjugation involves the following steps:
-Pilus formation: The donor cells (F+ cells) form a sex pilus and begin contact with recipient
cells (F- cell).
-Physical contact between donor and recipient cells: The pilus forms a conjugation tube and
enables direct contact between the donor and the recipient cells.
-Transfer of F-plasmid: The F-factor opens at the origin of replication. One strand is cut at the
origin of replication, and the 5' end enters the recipient cell.
-Synthesis of complementary strand: The donor cell then makes a single-stranded copy of its F-
plasmid and transmits/transfers it to recipient cell via sex pilus. The F plasmid becomes
integrated into the genome of recipient cell leading to the formation of a complementary strand.
The recipient cell now contains a copy of F plasmid and becomes a donor cell— capable of
producing a sex pilus and conjugate with other cells—transfer its DNA to other cells.
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3): Bacterial Transduction:
-The transfer of a portion of the DNA from one bacterium to another by a bacteriophage
(phage) is known as transduction. Bacteriophages are viruses that parasitize (or infect)
bacteria and use their machinery for their own replication. Structurally, bacteriophages consist
of a nucleic acid core [DNA or RNA (usually not both)] and a protein coat. Such viral particles
carrying bacterial DNA are known as transducing particles. Most bacteriophages carry their
genetic information (the phage genome) as a length of double-stranded DNA coiled up inside a
protein coat. [Look at the structural composition of bacteriophages at your revision time].
When bacteriophages multiply inside an infected bacterial cell (through the process of
replication), each phage head (or capsid) is normally filled with a copy of the replicated phage
genome. During the assembly of bacteriophage progeny inside infected bacteria, ‘packaging
errors’ may occur occasionally. A phage particle may have at its core (head) a
segment/fragment of the host DNA (genome/chromosome) besides its own nucleic acid. When
this phage particle infects another bacterium, the bacterial chromosome in the phage also gets
transferred to the new bacterium . This fragment may undergo recombination with the host
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bacterium’s chromosome and confer new property (or characteristic) to the (recipient)
bacterium. Thus, bacterial genes have been transduced by the phage into the second bacterial
cell.
-Although each virus has unique aspects to its life cycle, a general pattern of replication is
observable. The typical virus life cycle consists of five steps, namely (1) attachment to the host
cell, (2) entry into the host cell, (3) synthesis of viral nucleic acid and proteins within the host
cell, (4) self-assembly of virions (progeny virus) within the host cell, and (5) release and
maturation of virions from the host cell.
Types of Transduction:
-Two major types of transduction are known to occur in bacteria: generalized transduction and
specialized transduction.
A. Generalized transduction: Since phages of this type pick up any portion of the bacterial
chromosome at random, they are known as generalized transducing phages (i.e. this
involves any segment of the donor chromosome—or—random fragments of disintegrating
host DNA are picked up by the phage during assembly; any gene can be transmitted this
way). Transduction of cellular DNA by a virus can lead to recombination between the DNA
of the donor host cell and the DNA of the recipient host cell. A bacteriophage genome that
has been integrated into chromosome of a host cell is known as a prophage. The process of
generalized transduction is typical of bacteriophages. Genes can be transduced only
between fairly closely related strains as particular phages usually attack only a limited range
of bacteria. As well as chromosomal genes, generalized transducing bacteriophages may
also pick up and transfer plasmid DNA. As an example, the penicillinase gene in
staphylococci is usually located on a plasmid, and it may be transferred into other
staphylococcal strains by transduction.
-The stages/steps involved in generalized transduction include :
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1) Firstly, the bacteriophage attacks the bacterial cell (donor cell).
2) The bacteriophage injects its genetic material, which then penetrates the bacterial
chromosome.
3) The phage DNA then controls the host cell machinery. This cause the degradation of the
bacterial chromosomal DNA, which results in small fragments of the genetic material.
4) The replication of phage DNA occurs. At the time of packaging, the genetic material of
the bacterial DNA accidentally gets into the bacteriophages by a phenomenon called
“false packaging”.
5) Then, the biosynthesis of bacteriophages occurs, and the phages get to assemble and
finally cause lysis of the donor cell.
6) The bacteriophages then attack the other bacterial cell (recipient cell), and again it will
penetrate its phage DNA into the host cell.
7) The phage DNA will incorporate in the recipient’s DNA and leads to the formation of a
recombinant cell.
B. Specialized (specific) or restricted transduction: In specialized or restricted transduction, a
specific bacteriophage transduces only a particular genetic trait (i.e. only specific sections
of host DNA (genome) can be transferred to recipient bacteria or—only a few specific donor
genes can be incorporated into the virus). Restricted transduction has been studied
intensively in the ‘lambda’ phage of E. coli. The prophage lambda is inserted in the bacterial
chromosome only between the genes determining galactose utilization (gal) and biotin
synthesis (bio) and therefore it transduces only either of these.
-The stages/steps involved in specialized transduction include :
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1) First, the bacteriophage attacks the bacterial cell or donor cell.
2) Then, the genetic material of the bacteriophage will penetrate the bacterial
chromosome.
3) The phage DNA will incorporate within the bacterial DNA and then it will start the
replication cycle.
4) The phage DNA then control over the cell machinery of the bacteria. This leads to the
assembly of the bacteriophages that consist of both phage and donor’s DNA.
5) After that, exposure to stimulus such as chemicals or a UV-light induces cell lysis and
results in the release of lysogens (i.e. lysogens are bacteria harboring a prophage).
6) Then, the bacteriophages attack the other bacterial cell (recipient cell). This
bacteriophage will penetrate the prophage DNA into the recipient cell.
7) The phage DNA and the donor cell DNA will incorporate in the recipient’s DNA,
which leads to the formation of the recombinant cell.
Applications/Role of Bacterial Transduction:
1. Gene therapy: bacterial transduction has huge potential of treating genetic diseases through
gene therapy.
2. Genetic (gene) mapping of bacteria: It provides an excellent tool for the (genetic) mapping
of bacterial genes.
3. Transduction is used to insert the genes of choices in animals and plant cells to modify the
genetic constituents and get the desired characteristics.
4. Contributes to the increase of genetic diversity: The process of bacterial transduction
generally increases genetic diversity of organisms. This is very important as it allows
organisms to adapt to different environmental changes.
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5. Serves as a key to antibiotic resistance: The plasmids determining antibiotic resistance in
bacteria such as Staphylococci are transferred from cell to cell by transduction.
6. Generalized transduction has been used in mutagenesis, transferring plasmids and
transposons, and determining whether different genera of bacteria have homologous genes.
Lysogenic Conversion:
-Bacteriophages exhibit two types of life cycle.
(1): Virulent or lytic cycle; (2): Temperate or nonlytic or lysogenic cycle:
i. Virulent or lytic cycle : In the virulent or lytic cycle, large numbers of progeny phages
are built up inside the host bacterium, which ruptures to release them. The release of
progeny phages kills the parasitized bacterial (host) cell. Lytic cycle occurs during
generalized transduction of a virulent phage.
ii. Temperate or nonlytic cycle : In the temperate or nonlytic cycle, the host bacterium is
unharmed. The phage DNA becomes integrated with the bacterial chromosome as the
prophage, and is replicated stably as part of the host cell chromosome and is transferred
to the daughter cells. This process is called lysogeny and bacteria harbouring prophages
are called lysogenic bacteria. In lysogenic bacteria, the prophage behaves as an
additional segment of the bacterial chromosome, coding for new characteristic. This
process by which the prophage DNA confers genetic information to a bacterium is
called lysogenic or phage conversion. In transduction, the phage acts only as a vehicle
carrying bacterial genes from one cell to another but in lysogenic conversion the phage
DNA itself is the new genetic element.
Lysogenic conversion influences susceptibility to bacteriophages (immunity to
superinfection with the same or related phages) and antigenic characteristics.
Lysogeny is extremely frequent in nature. This is a symbiotic relationship in which
the integrated phage DNA imparts immunity to the lysogenized cell against
superinfection by genetically related phages, as well as certain unrelated phages.
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Biomedical Applications of Bacteriophages:
-Phage therapy has become a potential therapeutic option. For instance, bacteriophage therapy
has been used in treatment of various bacterial pathogens (e.g. streptococcal and staphylococcal
infections), and antibiotic-resistance bacteria (like Salmonella spp. Clostridium difficile, and
diarrheagenic E. coli).
-Phage-based medical products (phage cocktails) have been developed in the commercial
laboratory for human use. These products are either in the form of a lysate in broth cultures or
in the form of a water soluble jelly against targeted bacteria like, Staphylococci spp.,
Streptococci spp., and E. coli. They have been used in treating abscesses, burn injuries, open
septic wounds and vaginitis, mastoid infections, and respiratory tract infections. For examples
“phagoburn”— which contains bacteriophage cocktail targeting pathogenic strain of E. coli and
Pseudomonas aeruginosa and its infection in serious burn patients.
-Phage therapy is an important alternative to overcome critical limitations of antibiotic therapy
due to emergence of bacterial resistance, like, in the case of Clostridium difficile infection.
-Development of phage bioderm is another important area of application in clinical sector. Phage
bioderm is a therapeutic auto-degrading, non-toxic, biopolymer complex containing phages that
help in healing of wounds and burns, osteomyelitis, and periodontal diseases.
-The specificity of the interaction of a phage with its host cell immediately lends itself to
methods for the detection of pathogenic bacteria, through phage typing—where a panel of
phages with different lytic spectra is used to discriminate (or identify and distinguish) between
different isolates of a bacterial species or genus, according to their ability to infect the isolate
and form plaques (a plaque is a clear area which results from the lysis of bacteria).
-Phage sensitivity and specificity have been explored in designing phage-based biosensors—
which could be an improved alternative to antibody based immunoassay techniques like ELISA.
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-Cellular proteins and peptides, antibodies (or their fragments), and antigen molecules have been
expressed on the surfaces of phages for developing pathogen detection biosensors, molecular
imaging, and gene delivery.
TOPIC: 11
MUTATION:
- Mutation is a random, undirected, heritable variation or change caused by an alteration in
the nucleotide sequence at some point of the DNA of the cell. It may be due to addition,
deletion or substitution of one or more. Mutations are usually detrimental, but they can also
lead to beneficial changes.
-A strain carrying such a change (i.e. mutation) is known as a mutant. A mutant by definition
differs from its parental strain in genotype—the nucleotide sequence of the genome. But in
addition, the observable properties of the mutant (i.e. its phenotype) may also be altered
relative to the parental strain. One refers to this altered phenotype as a mutant phenotype. It is
common to refer to a strain isolated from nature as a wild-type strain (i.e. wild-type is a
reference organism, usually found naturally).
-The cornerstone of bacterial genetics has been, until recently, the isolation of specific mutants,
i.e. strains in which the gene concerned is altered. This alteration shows up as a change in the
corresponding characteristics of the organism. It is this change in the observable properties of
the organism (the phenotype) that is used to follow the transmission of the gene. The genetic
nature of the organism (the genotype) is inferred from the observable characteristics (i.e.
physiological, physical or phenotypic variations). The key difference between physiological
and genetic variation is whether the altered characteristic of an organism can be inherited.
- Genotype: genetic constitution of a cell that is transmitted to its progeny.
- Phenotype: physical expression of the genotype in a given environment (i.e. characteristics
displayed by the organism).
- Variations (two types) :
1. Phenotypic variations:
Influenced by the environment; Temporary and not heritable.
2. Genotypic variations:
Not influenced by the environment; Stable and heritable.
Types of Mutation:
-Mutations can be divided conveniently into: (i) spontaneous mutations; (ii) induced mutation;
and (iii) point mutations.
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A. Spontaneous mutations : Many mutations occur spontaneously in nature and these
spontaneous mutations apparently occur in the absence of any added mutation-causing
agents (or mutagens).
They occur naturally without human interference; are unpredictable and have no known
causes. Spontaneous mutations, also known as background mutation can however, occur
as a result of exposure to natural radiation (heat, cosmic rays, UV rays etc.) that alters
the structure of bases in the DNA. Also, oxygen radicals can affect DNA structure by
chemically modifying DNA. Normal cell activities such as mistakes or errors during
DNA replication can also lead to spontaneous mutations.
B. Induced mutations : These mutations occur as a result of exposure of the organism or cells
to some physical or chemical agent known as mutagens. In other words, induced mutation
is an artificial kind of mutation.
A mutagen is a physical or chemical agent that changes the genetic material, usually
DNA of an organism and thus increases the frequency of mutations above the natural
background level.
Types of Mutagens :
1. Physical agents : (i) UV rays; (ii) Ionizing radiation, e.g. X-rays; (iii) Visible light;
(iv) Heat.
2. Chemical agents : (i) Alkylating agents; (ii) Acridine dyes; (iii) 5-Bromouracil; (iv)
2aminopurine; Nitrous acid.
3. Biological agents: (i) Virus; (ii) Bacteria; (iii) Transposable genetic elements.
C. Point mutations : As the name suggests, point mutations affect just one point (base pair) in
a gene. Such mutations may be a change to or substitution of a different base pair.
Alternatively, a point mutation can result in the deletion or addition of a base pair. Point
mutations are the most common mechanism of mutation. In general, they are reversible and
are of two classes:
1. Base pair substitution: This comprises those mutants in which a single base pair
(nucleotide) has been substituted for another pair, and can be subdivided into transition
(one purine e.g. A or G is replaced by other purine or a pyrimidine e.g. T or C is
replaced by other pyrimidine) and tranversion (substitution of a purine for a pyrimidine
and vice versa in base pairing).
Depending on the placement of the substituted base when the mRNA is translated, this
may cause no change (silent mutation), lead to the insertion of the wrong amino acid
(missense mutation) or generate a stop codon (nonsense mutation), prematurely
terminating the polypeptide.
Silent mutation : type of point mutation that code for the same amino acid—thus, has no
effect on amino acid sequence—no effect on the protein’s structure—resulting in an
unaltered protein.
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Examples of silent mutations:
-CCA (Proline) → CCG (Proline) → CCU(Proline) → CCC (Proline) - AAA (Lysine) → AAG
(Lysine).
-CAG (Glutamine) → CAA (Glutamine).
Missense mutation: missense mutation occurs when the triplet code is altered so as to
specify an amino acid different from that normally located at a particular position in the
protein. [ i.e. a missense mutation: a single substitution mutation which results in one
wrong codon and, therefore, one wrong amino acid].
Examples of missense mutations:
-AAA Lysine) → GAA (Glutamine)
-AGG (Arginine) → ACG (Threonine)
-CAG (Glutamine) → CAT (Histidine)
Nonsense mutation: Deletion of a nucleotide within a gene may cause premature
polypeptide chain termination by generating a nonsense codon (UAG, UAA or UGA).
This is known as nonsense mutation. [i.e. a nonsense mutation: a single substitution
mutation which results in transcription of a stop or nonsense codon resulting in the
termination of polypeptide chain].
Examples of nonsense mutations:
-CAA (Glutamine) → TAA (stop codon)
-CAG (Glutamine) → UAG (stop codon)
2. Base pair deletion or insertion:
Frameshift mutations: Also known as a reading frame shift, is a genetic mutation
caused by indels (insertion or deletion) of a certain number of nucleotides (bases) in
a DNA sequence.
- If the number of bases inserted or deleted is not a multiple of three, there will be shift
in the reading frame, i.e. frameshift mutations.
[A frameshift mutation in a gene refers to the insertion or deletion of nucleotide
bases in numbers that are not multiples of three].
-This shifts the normal ‘reading frame’ of the coded message forming newest of triplet codon.
The coded message is read correctly up to the point of addition or deletion, but the subsequent
codons will specify the incorrect amino acids.
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- CGA (Arginine) → UGA (stop codon)
-In other words, frame shift mutation causes a shift in open reading frame and all codons and
amino acids downstream from that mutation (or change) are usually wrong.
-In case one of the wrong codons turns out to be a stop or nonsense codon, the protein is
terminated at that point.
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Other Types of Mutations:
- Lethal mutation : Sometimes some mutations affect vital functions and the bacterial cell
become nonviable. Hence, those mutations that can kill the cell are called lethal mutation.
- Conditional lethal mutation : Sometimes a mutation may affect an organism in such a way that
the mutant can survive only in certain environmental condition. Example; a temperature
sensitive mutant can survive at permissive temperature of 35°C but not at restrictive
temperature of 39°C.
- Suppressor mutation : is a (second) mutation that counters (or mask) the phenotypic effect of a
previous (or an earlier) mutation. It may either alleviate or revert the phenotypic effects of an
already existing mutation. Genetic suppression therefore restores the phenotype seen prior to
the original background mutation.
- Recognition of mutation : Mutation can be best recognized when it involves a function which
can be readily observed by experimental methods like alteration in colonial morphology,
pigmentation, alteration in cell surface antigens, sensitivity to bacteriophages or bacteriocins,
loss of ability to produce capsule or flagella, loss of virulence and change in biochemical
characters.
Importance of Bacterial Mutations:
1. Drug resistance and development of live vaccines : The practical importance of bacterial
mutations is mainly in the field of drug resistance and development of live vaccines. Some
organisms have been sub-cultured in the laboratory for many generations until they lost
their virulence for man (e.g. BCG vaccine). This is known as live attenuated vaccine.
2. Mutations are the ultimate source of variation : Some mutations lead to newer versions of
proteins and help the organisms to adapt to changes in the environment.
3. Mutations are the origin or foundation of evolution : They are the source of variability,
which increases the adaptability of an organism to its environment and wards off
deterioration in unfavorable conditions.
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