COURSE CODE: BIT18R271

COURSE NAME: MICROBIOLOGY

UNIT 3 - BACTERIAL PHYSIOLOGY AND GENETICS
Syllabus:
Lesson 1. Nutritional requirement and cultivation of bacteria
Lesson 2. Bacterial growth curve
Lesson 3. Biosynthesis and transport of molecules
Lesson 4. Genetic exchange in bacteria
Lesson 5. Mutation and recombination

Course Outcomes:

CO 1. Describe diversity, classification and identification methods of microorganisms.

CO 2. Explain the structure and function of components of bacterial cell, fungi,
viruses and algae.
CO 3. Explain the physiology of bacteria and genetic exchange in bacteria.
CO 4. Understand the mechanism of pathogenesis of various infectious diseases
and ways of preventing / controlling them.
CO 5. Demonstrate the knowledge as to how microorganisms interact with their
environment
 Lesson 1: NUTRITIONAL REQUIREMENTS OF BACTERIA:

• Nutrients are materials that are acquired from the environment and are used for
growth and metabolism. Microorganisms (or microbes) vary significantly in the
source, chemical form, and amount they will need of these essential elements.
• Macro-nutrients are needed in large amounts and micro-nutrients are needed in
trace or small amounts.

Sources of Essential Nutrients for Microorganisms

• Nutrients are materials that are acquired from the environment and are used for
growth and metabolism.
• Microorganisms vary significantly in terms of the source, chemical form, and
amount of essential elements they need. Some examples of these essential
nutrients are carbon, oxygen, hydrogen, phosphorus, and sulfur.
• There are two categories of essential nutrients: macro-nutrients (which are
needed in large amounts) and micro-nutrients (which are needed in trace or
small amounts). Macro-nutrients usually help maintain the cell structure and
metabolism. Micro-nutrients help enzyme function and maintain protein
structure.
Organic and Inorganic Nutrients
• Organic nutrients contain some combination of carbon and hydrogen atoms.
Inorganic nutrients are elements or simply molecules that are made of elements
other than carbon and hydrogen.
Essential Nutrients

• The sources of common essential nutrients are carbon, hydrogen, nitrogen,

oxygen, phosphorus, and sulfur.
 • Organisms usually absorb carbon when it is in its organic form. Carbon in its
organic form is usually a product of living things. Another essential nutrient,
nitrogen, is part of the structure of protein, DNA, RNA, and ATP. Nitrogen is
important for heterotroph survival, but it must first be degraded into basic
building blocks, such as amino acids, in order to be used. Oxygen is an
important component of both organic and inorganic compounds. It is essential to
the metabolism of many organisms.
• Hydrogen has many important jobs including maintaining the pH of solutions
and providing free energy in reactions of respiration. Phosphate is an important
player in making nucleic acids and cellular energy transfers. Without sufficient
phosphate, an organism will cease to grow. Lastly, sulfur is found in rocks and
sediments and is found widely in mineral form.

The Role of Nutrients

• Nutrients are necessary for microbial growth and play a vital role in the proper
cultivation of microorganisms in the laboratory and for proper growth in their
natural environments.
• The types of nutrients that are required include those that supply energy, carbon
and additional necessary materials. The nutrients used to propagate growth are
organism specific, based on their cellular and metabolic processes.
• The common nutrients which are found to be required in all living things
include carbon, nitrogen, sulfur, phosphorus, potassium, magnesium, calcium,
oxygen, iron and additional trace elements. Essential nutrients are nutrients
absolutely required by an organism.
• Two categories of essential nutrients are macro- and micro-nutrients.
Macronutrients are necessary in large amounts; micronutrients tend to be needed
in smaller amounts and are often trace elements.
• All organisms require a source of energy. Some rely on chemical compounds
for their energy and are designated as chemotrophs. Others can utilize radiant
energy (light) and are called phototrophs. Both chemotrophs and phototrophs
exist among bacteria
• All organisms require a source of electrons for their metabolism. Some
organisms can use reduced inorganic compounds as electron donors and are
termed lithotrophs (some may be chemolithotrophs, others photolithotrophs)
Other organisms use organic compounds as electron donors and are called
organotrophs (some are chemoorganotrophs, others photo organotrophs)
• All organisms require carbon in some form for use in synthesizing cell
components. All organisms require at least small amounts of CO 2. However,
some can use CO2 as their major or even sole source of carbon; such organisms
are termed autotrophs. Others require organic compounds as their carbon source
and are termed heterotrophs.
• All organisms require nitrogen in some form for cell components. Bacteria are
extremely versatile in this respect. Unlike eukaryotes, some bacteria can use
atmospheric nitrogen. Others thrive on inorganic nitrogen compounds such as
nitrates, nitrites, or ammonium salts, and still others derive nitrogen from
organic compounds such as amino acids.
• All organisms require oxygen , Sulfur and phosphorus for cell components.
Oxygen is provided in various forms, such as water; component atoms of
various nutrients; or molecular oxygen. Sulfur is needed for synthesis of certain
amino acids (cysteine, cystine, and methionine). Some bacteria require organic
 sulfur compounds, some are capable of utilizing inorganic sulfur compounds,
and some can even use elemental sulfur. Phosphorus, usually supplied in the
form of phosphate, is an essential component of nucleotides, nucleic acids,
phospholipids, teichoic acids and other compounds.
• All living organisms require metal ions, such as K, Ca2 , Mg2 , and Fe2 for
normal growth. Other metal ions are also needed but usually only at very low
concentrations, such as Zn2+, Cu, Mn2 +, Mo, Ni, B, and Co these are often
termed trace elements and often occur as contaminants of other components of
culture media in amounts sufficient to support bacterial growth.
• All living organisms contain vitamins and vitamin like compounds. These
function either as coenzymes for several enzymes or as the building blocks for
coenzymes. Some bacteria are capable of synthesizing their entire requirement
of vitamins from other compounds in the culture medium,
• All living organisms require water, and in the case of bacteria all nutrients must
be in aqueous solution before they can enter the cells. Water is a highly polar
compound that is unequaled in its ability to dissolve or disperse cellular
components and to provide a suitable milieu for the various metabolic reactions
of a cell.

Nutrients as Limiting Factors

• In regard to required nutrients for proper growth, there are often limiting factors
involved. The limiting factor or limiting nutrient affects and controls growth.
• The availability of specific nutrients dictates organismal growth by controlling
and limiting activation of cellular and metabolic pathways necessary for
progress.
 • When all nutrients and parameters are ideal and constant during the growth
phase, this is regarded as a steady state: all requirements are present and
microorganisms thrive. In circumstances where there are less than ideal
parameters, such as a lack of specific requirements, the growth process is
affected.

Figure 1: Nutritional requirements of Microorganisms

Culture Media

• Culture medium or growth medium is a liquid or gel designed to support the

growth of microorganisms. Many special-purpose media are needed to facilitate
recognition, enumeration, and isolation of certain types of bacteria. To meet
these needs, the microbiologist has available numerous media which, on the
basis of their application or function
 • Chemically defined media are needed for the cultivation of autotrophs and
media also useful for defining the nutritional requirements of heterotrophs.
However, for the routine cultivation of heterotrophs, chemically defined media
are not generally used.
• Instead, certain complex raw materials such as peptones, meat extract, and yeast
extract are used, and the resulting media support the growth of a wide variety of
heterotrophic bacteria. Agar is included as a non-nutritive solidifying agent
when a solid medium is desired.
• Examples of relatively simple liquid and solid media that support the growth of
many common heterotrophs are nutrient broth and nutrient agar. The addition of
yeast extract to each of these formulas improves the nutrient quality, since yeast
extract contains several of the B vitamins and other growth-promoting
substances.
• Other complex supplements such as bovine rumen fluid, animal blood, blood
serum, or extracts of plant and animal tissues may be required for the cultivation
of certain fastidious heterotrophs.

Nutrient Broths and Agar Plates

• These are the most common growth media, although specialized media are
sometimes required for microorganism and cell culture growth. Some
organisms, termed fastidious organisms, need specialized environments due to
complex nutritional requirements. Viruses, for example, are obligate
intracellular parasites and require a growth medium containing living cells.
Many human microbial pathogens also require the use of human cells or cell
lysates to grow on a media.
 • The most common growth media nutrient broths (liquid nutrient medium) or LB
medium (Lysogeny Broth) are liquid. These are often mixed with agar and
poured into Petri dishes to solidify. These agar plates provide a solid medium on
which microbes may be cultured. They remain solid, as very few bacteria are
able to decompose agar. Many microbes can also be grown in liquid cultures
comprised of liquid nutrient media without agar.

Defined Vs Undefined media

• This is an important distinction between growth media types. A defined medium

will have known quantities of all ingredients. For microorganisms, it provides
trace elements and vitamins required by the microbe and especially a defined
carbon and nitrogen source.
• Glucose or glycerol are often used as carbon sources, and ammonium salts or
nitrates as inorganic nitrogen sources. An undefined medium has some complex
ingredients, such as yeast extract, which consists of a mixture of many, many
chemical species in unknown proportions.
• Undefined media are sometimes chosen based on price and sometimes by
necessity – some microorganisms have never been cultured on defined media.
• There are many different types of media that can be used to grow specific
microbes, and even promote certain cellular processes; such as wort, the
medium which is the growth media for the yeast that makes beer.
• Without wort in certain conditions, fermentation cannot occur and the beer will
not contain alcohol or be carbonated (bubbly).
 Figure 2: Examples of defined media

Defined Culture Media

• Nutrient media – A source of amino acids and nitrogen (e.g., beef, yeast
extract). This is an undefined medium because the amino acid source contains a
variety of compounds with the exact composition being unknown. These media
contain all the elements that most bacteria need for growth and are non-
selective, so they are used for the general cultivation and maintenance of
bacteria kept in laboratory-culture collections.
 • Minimal media – Media that contains the minimum nutrients possible for
colony growth, generally without the presence of amino acids, and are often
used by microbiologists and geneticists to grow “wild type” microorganisms.
These media can also be used to select for or against the growth of specific
microbes. Usually, a fair amount of information must be known about the
microbe to determine its minimal media requirements.
• Selective media – Used for the growth of only selected microorganisms. For
example, if a microorganism is resistant to a certain antibiotic, such as
ampicillin or tetracycline, then that antibiotic can be added to the medium in
order to prevent other cells, which do not possess the resistance, from growing.
• Differential media – Also known as indicator media, are used to distinguish
one microorganism type from another growing on the same media. This type of
media uses the biochemical characteristics of a microorganism growing in the
presence of specific nutrients or indicators (such as neutral red, phenol red,
eosin y, or methylene blue) added to the medium to visibly indicate the defining
characteristics of a microorganism. This type of media is used for the detection
and identification of microorganisms.

These few examples of general media types provide some indication only; there are a
myriad of different types of media that can be used to grow and control microbes.

Complex and Synthetic Media

• A chemically defined medium is entirely free of animal-derived components

(including microbial derived components such as yeast extract) and represents
the purest and most consistent cell culture environment. By definition
chemically defined media cannot contain either fetal bovine serum, bovine
 serum albumin, or human serum albumin as these products are derived from
bovine or human sources and contain complex mixes of albumins and lipids.
• In defined media all the chemical compounds are known, while undefined
media has partially unknown chemical constituents.
• There are many types of culture media, which is food that microbes can live on.
Two major sub types of media are complex and synthetic medias, known as
undefined and defined media.
• An undefined medium has some complex ingredients, such as yeast extract or
casein hydrolysate, which consist of a mixture of many, many chemical species
in unknown proportions.
• A defined medium (also known as chemically defined medium or synthetic
medium) is a medium in which all the chemicals used are known, no yeast,
animal, or plant tissue is present. A chemically defined medium is a growth
medium suitable for the culture of microbes or animal cells (including human)
of which all of the chemical components are known.

Figure 3: Common complex media

Selective and Differential Media

• Selective media allows for the growth of specific organisms, while differential
media is used to distinguish one organism from another.
• There are many types of media used in the studies of microbes. Two types of
media with similar implying names but very different functions, referred to as
selective and differential media, are defined as follows.
• Selective media are used for the growth of only selected microorganisms. For
example, if a microorganism is resistant to a certain antibiotic, such as
ampicillin or tetracycline, then that antibiotic can be added to the medium in
order to prevent other cells, which do not possess the resistance, from growing.

Figure 4: Synthetic and Differential media

• Normally, the presence of a specific gene or an allele of a gene confers upon the
cell the ability to grow in the selective medium. In such cases, the gene is
termed a marker. Selective growth media for eukaryotic cells commonly contain
neomycin to select cells that have been successfully transfected with a plasmid
carrying the neomycin resistance gene as a marker. Gancyclovir is an exception
to the rule as it is used to specifically kill cells that carry its respective marker,
the Herpes simplex virus thymidine kinase (HSV TK). Some examples of
selective media include:
• Eosin methylene blue (EMB) that contains methylene blue – toxic to Gram-
positive bacteria, allowing only the growth of Gram negative bacteria.
✓ YM (yeast and mold) which has a low pH, deterring bacterial growth.
✓ MacConkey agar for Gram-negative bacteria.
✓ Hektoen enteric agar (HE) which is selective for Gram-negative bacteria.
✓ Mannitol salt agar (MSA) which is selective for Gram-positive bacteria and
differential for mannitol.
✓ Terrific Broth (TB) is used with glycerol in cultivating recombinant strains of
Escherichia coli.
✓ Xylose lysine deoxycholate (XLD), which is selective for Gram-negative
bacteria buffered charcoal yeast extract agar, which is selective for certain
gram-negative bacteria, especially Legionella pneumophila.
• Differential media or indicator media distinguish one microorganism type from
another growing on the same media. This type of media uses the biochemical
characteristics of a microorganism growing in the presence of specific nutrients
or indicators (such as neutral red, phenol red, eosin or methylene blue) added to
the medium to visibly indicate the defining characteristics of a microorganism.
• This type of media is used for the detection of microorganisms and by
molecular biologists to detect recombinant strains of bacteria.
 Examples of differential media include: Blood agar (used in strep tests), which
contains bovine heart blood that becomes transparent in the presence of
hemolytic.

✓ Streptococcus eosin methylene blue (EMB), which is differential for lactose and
sucrose fermentation.

✓ MacConkey (MCK), which is differential for lactose fermentation mannitol salt

agar (MSA), which is differential for mannitol fermentation.

✓ X-gal plates, which are differential for lac operon mutants.

Lesson 2: BACTERIAL GROWTH CURVE

• The most common means of bacterial reproduction is binary fission; one cell
divides, producing two cells. Thus, if we start with a single bacterium, the
increase in population is by geometric progression:
1 → 2 → 22 → 23→ 24……... → 2n
n = number of generations
• When a broth culture is inoculated with a small bacterial inoculum, the
population size of the bacteria increases showing a classical pattern. When
plotted on a graph, a distinct curve is obtained referred to as the bacterial growth
curve.

Method of Obtaining Bacterial Growth Curve:

• A population growth curve for any particular species of bacterium may be

determined by growing a pure culture of the organism in a liquid medium at a
constant temperature.
 • Samples of the culture are collected at fixed intervals (e.g., every 30 minutes),
and the number of viable organisms in each sample is determined.
• The data are then plotted on logarithmic graph paper.
• The logarithm of the number of bacteria per milliliter of medium is plotted
against time.

Figure 5: Growth curve

The bacterial growth curve shows the following four distinct phases:
1. Lag phase:
• After a liquid culture broth is inoculated, the multiplication of bacteria does not
start immediately. It takes some time to multiply.
• The time between inoculation and beginning of multiplication is known as lag
phase.
• In this phase, the inoculated bacteria become acclimatized to the environment,
switch on various enzymes, and adjust to the environmental temperature and
atmospheric conditions.
• During this phase, there is an increase in size of bacteria but no appreciable
increase in number of bacterial cells. The cells are active metabolically.
• The duration of the lag phase varies with the bacterial species, nature of culture
medium, incubation temperature, etc.
• It may vary from 1 hour to several days.
2. Log phase:
• This phase is characterized by rapid exponential cell growth (i.e., 1 to 2 to 4 to 8
and so on).
• The bacterial population doubles during every generation. They multiply at their
maximum rate.
• The bacterial cells are small and uniformly stained.
• The microbes are sensitive to adverse conditions, such as antibiotics and other
antimicrobial agents.
• Growth rate is the greatest during the log phase.
• The log phase is always brief, unless the rapidly dividing culture is maintained
by constant addition of nutrients and frequent removal of waste products.
• When plotted on logarithmic graph paper, the log phase appears as a steeply
sloped straight line.
3. Stationary phase:
• After log phase, the bacterial growth almost stops completely due to lack of
essential nutrients, lack of water oxygen, change in pH of the medium, etc. and
accumulation of their own toxic metabolic wastes.
 • It is during this phase that the culture is at its greatest population density.
• However, Death rate of bacteria exceeds the rate of replication of bacteria.
• Endospores start forming during this stage.
• Bacteria become Gram variable and show irregular staining.
• Many bacteria start producing exotoxins.
4. Decline phase:
• During this phase, the bacterial population declines due to death of cells.
• The decline phase starts due to:
(a) accumulation of toxic products and autolytic enzymes and
(b) exhaustion of nutrients.
• Involution forms are common in this stage. Some cells assume various shapes,
becoming long, filamentous rods or branching or globular forms that are difficult
to identify.
• Some develop without a cell wall and are referred to as protoplasts,
spheroplasts, or L-phase variants (L-forms).
• When these involuted forms are inoculated into a fresh nutrient medium, they
usually revert to the original shape of the healthy bacteria.

Calculation of generation time:

• When growing exponentially by binary fission, the increase in a bacterial
population is by geometric progression. If we start with one cell, there are 2
cells in the first generation, 4 cells in the second generation, 8 cells in the third
generation, and so on. The generation time is the time interval required for the
cells (or population) to divide.
(G = t/n)
G (generation time) = (time, in minutes or hours)/n(number of generations)
t = time interval in hours or minutes
 B = number of bacteria at the beginning of a time interval
b = number of bacteria at the end of the time interval
n = number of generations
b = B x 2n (This equation is an expression of growth by binary fission)
Solve for n:
logb = logB + nlog2
n = logb-logB
log2
n = logb - logB
0.301
n = 3.3 logb/B
G = t/n
Solve for G
G= t
3.3 log b/B

What is the generation time of a bacterial population that increases from 10,000
cells to 10,000,000 cells in four hours of growth?

G= t_____
3.3 log b/B
G = 240 minutes
3.3 log 107/104

G = 240 minutes
3.3 x 3

G = 24 minutes

Significance of the Bacterial Growth Curve

• The study of bacterial growth curves is important when aiming to utilize or

inoculate known numbers of the bacterial isolate, for example to enhance plant
growth, increase biodegradation of toxic organics, or produce antibiotics or other
natural products at an industrial scale.
• Knowledge of bacterial growth kinetics and bacterial numbers in a culture medium
is important from both a research and commercial point of view.
• Growth kinetics is also useful for assessing whether particular strains of bacteria
are adapted to metabolize certain substrates, such as industrial waste or oil
pollution.
• Bacteria that are genetically engineered to clean up oil spills, for example, can be
grown in the presence of complex hydrocarbons to ensure that their growth would
not be repressed by the toxic effects of oil.
• Similarly, the slope and shape of growth curves produced from bacteria grown
with mixtures of industrial waste products can inform scientists whether the
bacteria can metabolize the particular substance, and how many potential energy
sources for the bacteria can be found in the waste mixture.

Bacteria on the basis of their oxygen requirements can be classified broadly into
aerobic and anaerobic bacteria.
Aerobic bacteria:

They require oxygen for their growth. They may be:

• Obligate aerobes—which can grow only in the presence of oxygen (e.g., P.

aeruginosa).
• Facultative aerobes—which are ordinary aerobes but can also grow without
oxygen (e.g., E. coli). Most of the pathogenic bacteria are facultative aerobes.
• Microaerophilic bacteria—those bacteria that can grow in the presence of low
oxygen and in the presence of low (4%) concentration of carbon dioxide (e.g.,
Campylobacter jejuni).
• Some fermentative organisms (e.g., Lactobacillus plantarum) are aerotolerant
but do not contain the enzyme catalase or superoxide dismutase. Oxygen is not
reduced, and therefore hydrogen peroxide (H2O2) and nascent oxygen (O2) are
not produced.

Anaerobic bacteria:

• Obligate anaerobes are the bacteria that can grow only in the absence of oxygen
(e.g., Clostridium botulinum, Clostridium tetani, etc.).
• These bacteria lack superoxide dismutase and catalase; hence oxygen is lethal to
these organisms.
 Lesson 3: BIOSYNTHESIS AND TRANSPORT OF MOLECULES

Metabolism: Chemical reactions takes place inside the cell. Two types: Energy
synthesis and Energy utilization

Biosynthesis: Process in which substrates are converted to more complex products.

The products which are produced are necessary for cellular and metabolic processes
and essential for survival
Prerequisite for biosynthesis:
1. Precursor molecules
2. Chemical energy
3. Catalytic enzymes
• Energy is mostly stored as ATP and managed in the form of chemical reactions
that involve the making and breaking of bonds and the transfer of electrons.
Exergonic reactions - release energy, making it available for cellular work.
Endergonic reactions are driven forward with the addition of energy.
• Some specialized enzyme systems are involved in the process to trap the energy
and produce products
Oxidation: loss of electrons
• An oxidizing agent (oxidant) will absorb electrons and will therefore become
reduced.
Example:
• The ferric ion is an oxidizing agent; it absorbs electrons and becomes reduced to
ferrous ion:
Fe 3+ + e - = Fe 2+
Ferric ion Electron Ferrous ion
Reduction: gain of electrons
• A reducing agent (reductant) donates electrons, becoming oxidized in the
process.
Example:
• The ferrous ion is a reducing agent, it donates electrons and becomes oxidized
to ferric ion:
Fe 2+ = Fe 3+ + e–
Ferrous ion Ferric ion Electron
ATP (Adenosine triphosphate):

• Energy-carrying molecule found in the cells of all living things.

• Also called Universal Energy Currency of cell
• ATP captures chemical energy obtained from the breakdown of food molecules
and releases it to fuel other cellular processes.
• Living organisms generate ATP through respiration and subsequently utilize
ATP to carry out cellular functions that are necessary for their survival, growth
and replication.

Figure 6: ATP Structure

Cellular Respiration - Production of ATP (Adenosine tri-phosphate) whose
molecules are oxidized to produce energy
▪ Aerobic respiration:
1. Occurs in bacteria and live in the presence of oxygen.
2. Final electron acceptor in reaction is oxygen
▪ Anaerobic respiration:
1. Occurs in bacteria and live in the environment where oxygen does not exist
2. Final electron acceptor is inorganic molecule
Glucose
• Important carbohydrate and considered as energy currency molecule
Glucose has major outcomes:
1. It may be stored as polysaccharide or sucrose
2. Glucose may be oxidized to a three carbon-compound through glycolysis
process to provide ATP and metabolic intermediates
3. Glucose may be oxidized through Pentose Phosphate Pathway to yield ribose 5-
phosphate for nucleic acid synthesis and NADPH for reductive biosynthetic
processes.
Overview of Aerobic metabolism

Three stages of aerobic metabolism

1. Glycolysis
2. Acetyl co A formation and TCA cycle
3. Electron transport chain or oxidative Phosphorylation
C6H12O6 (Glucose) + O2 (Oxygen) → CO2 (Carbon dioxide) + H2O (Water)
 Figure 7: Overall process

Figure 8: Overall process -Aerobic respiration

GLYCOLYSIS:
▪ Process in which the one glucose is broken down to produce energy
▪ Glucose is converted into pyruvate ATP, NADH and water
▪ Takes place in the cytoplasm
▪ Primary step of cellular respiration
▪ Also known as the EMP pathway (Embden–Meyerhof–Parnas).

Figure 9: Glycolysis process

Step 1: Uptake and Phosphorylation of Glucose by enzyme hexokinase

Step 2: Isomerization of Glucose 6 phosphate to Fructose 6 Phosphate by enzyme
Phospho hexoisomerase
Step 3: Phosphorylation of Fructose 6 Phosphate to Fructose 1,6 Bisphosphate by
enzyme Phosphofructokinase
Step 4: Cleavage of Fructose 1,6 Bisphosphate to Triose Phosphates
(Dihydroxyacetone and Glyceraldehyde 3 Phosphate) by the enzyme aldolase
Step 5: Interconversion of the Triose Phosphates by enzyme triose phosphate
isomerase
Step 6: Oxidation phosphorylation of GAP to 1,3 bisphosphoglycerate by the
enzyme glyceraldehyde 3 phosphate dehydrogenase
Step 7: Conversion of 1,3-Biphosphoglycerate to 3-phosphoglycerate by the
enzyme Phosphoglycerate kinase
Step 8: Conversion of 3-phosphoglycerate to 2-phosphoglycerate by the enzyme
Phosphoglycerate mutase
Step 9: Dehydration of 2-Phosphoglycerate to Phosphoenolpyruvate by the enzyme
Enolase
Step 10: Conversion of Phosphoenolpyruvate to Pyruvate by the enzyme Pyruvate
Kinase
Respiration vs Fermentation

Figure 10: Respiration vs Fermentation

Krebs cycle

▪ Also known as the TCA or citric acid cycle

▪ Main source of energy for cells and an important part of aerobic respiration.
▪ TCA cycle is part of the larger glucose metabolism whereby glucose is oxidized
to form pyruvate, which is then oxidized and enters the TCA cycle as acetyl-
CoA.

Figure 11: Krebs cycle

 Step 1: The TCA cycle begins with an enzymatic aldol addition reaction of
acetyl CoA to oxaloacetate, forming citrate.
Step 2: The citrate is isomerized by a dehydration-hydration sequence to yield
isocitrate.
Step 3: Further, enzymatic oxidation and decarboxylation gives 2-ketoglutarate.
Step 4: After another enzymatic decarboxylation and oxidation, 2-ketoglutarate
is transformed into succinyl-CoA.
Step 5: The hydrolysis of this metabolite to succinate is coupled to the
phosphorylation of guanosine diphosphate (GDP) to guanosine triphosphate
(GTP).
Step 6: Enzymatic desaturation by flavin adenine dinucleotide (FAD)-dependent
succinate dehydrogenase yields fumarate.
Step 7: After stereospecific hydration, fumarate catalysed by fumarase is
transformed to L-malate.
Step 8: The last step of NAD-coupled oxidation of L-malate to oxaloacetate is
catalysed by malate dehydrogenase and closes the cycle.

Oxidative Phosphorylation
▪ Series of proteins and organic molecules found in the inner membrane of the
mitochondria. Electrons are passed from one member of the transport chain to
another in a series of redox reactions.
▪ Process of shuttles electrons from NADH and FADH2 to molecular oxygen is
called Electron transport chain
▪ Energy released in these reactions is captured as a proton gradient, which is then
used to make ATP in a process called Chemiosmosis
▪ Electron transport chain and Chemiosmosis → Oxidative Phosphorylation
 Figure 12: ETC and Chemiosmosis

Step 1: Delivery of electrons by NADH and FADH

Step 2: Electron transfer and proton pumping.
Step 3: Splitting of oxygen to form water.
Step 4: Gradient-driven synthesis of ATP.

Overview of Anaerobic Metabolism

▪ Energy in glucose without the presence of oxygen.

▪ Most organisms require oxygen to produce ATP. Without oxygen, electron
transport chain cease to operate. In that case, glycolysis couples with
fermentation to produce ATP
▪ Lactic acid is the end product of anaerobic metabolism
Types of Fermentation

Figure 13: Fermentation types

Two common types

▪ Alcohol Fermentation – Pyruvate converted into Ethanol
▪ Lactic acid Fermentation – Pyruvate converted into Lactate
 Lesson 4 - GENETIC EXCHANGE IN BACTERIA
• Bacteria, being single-celled prokaryotic organisms, do not have a male or
female version. Bacteria reproduce asexually. In asexual reproduction, the
"parent" produces a genetically identical copy of itself.

Binary Fission
• Bacteria reproduce through a process called binary fission. Transverse binary
fission is an asexual reproductive process. During binary fission, the
chromosome copies itself, forming two genetically identical copies. Then, the
cell enlarges and divides into two new daughter cells. The two daughter cells
are identical to the parent cell. Binary fission can happen very rapidly.
Budding
• Some bacteria, such as Rhodopseudomonas acidophila, reproduce by budding,
process in which a small protuberance (bud) develops at one end of the cell;
this enlarges and eventually develops into a new cell which separates from the
parent. In some budding bacteria, such as Hyphomicrobium species, the bud
may develop at the end of a prostheca
Fragmentation
• Bacteria that produce extensive filamentous growth, such as Nocardia species,
reproduce by fragmentation of the filaments into small bacillary or coccoid
cells, each of which gives rise to new growth.
Formation of Conidiospores
• Species of the genus Streptomyces and related bacteria produce many spores or
Sporangiospores per organism by developing cross walls (septation) at the
hyphal tips; each spore gives rise to a new organism.
Exchanging DNA

Are the bacteria being male or female?

Absolutely, the answer is no gender. So, sexual reproduction does not occur in
bacteria. But not all new bacteria are clones. This is because bacteria can acquire new
DNA. This process occurs in three different ways:

Conjugation: In conjugation, DNA passes through an extension on the surface of one

bacterium and travels to another bacterium. Bacteria essential exchange DNA via
conjugation.

Transformation: In transformation, bacteria pick up pieces of DNA from their

environment.

Transduction: In transduction, viruses that infect bacteria carry DNA from one
bacterium to another.

BINARY FISSION

Binary fission may be as old as the very first forms of life – over 3.5 billion years old.
However, the process has remained unchanged ever since then. Read on to explore
binary fission in bacteria and amoeba reproduction in detail.

Definition

“Binary fission is a form of asexual reproduction in which an organism divides into

two, each part carrying one copy of genetic material.”
What is Binary Fission?

• Binary fission is a type of asexual reproduction typically observed in

prokaryotes and a few single-celled eukaryotes. In this method of asexual
reproduction, there is a separation of the parent cell into two new daughter
cells. This process happens with the division and duplication of the parent’s
genetic matter into two parts. Here, each daughter cell receives one copy of its
parent DNA.
• It is a primary method of reproduction in prokaryotic organisms. Binary Fission
occurs without any spindle apparatus formation in the cell. In this process, the
single DNA molecule begins replication and then attaches each copy to various
parts of the cell membrane.
• When the cell starts to get drawn apart, the original (actual) and replicated
chromosomes get apart.
• However, asexual mode of reproduction has a significant drawback. All
resultant cells are genetically identical, mirror copies of each other and the
parent cell. Most antibiotics work on this principle.
• If a parent cell is vulnerable to an antibiotic, then all resultant daughter cells are
vulnerable too. If a mutation occurs in their genes, then it can render a
particular strain resistant to antibiotics.
• Prokaryotes such as E. coli, Archaea as well as eukaryotes such as euglena
reproduce through binary fission.

Binary Fission in Bacteria

• The process of binary fission is usually rapid, and its speed varies among
species. The time required by bacteria to double the number of cells it has is
called doubling time. Furthermore, each species requires specific conditions for
 its growth. These conditions include pH levels, temperature, oxygen, light,
moisture, osmotic pressure.
• For instance, mesophiles thrive at moderate temperatures ranging from 20 °C to
45 °C. The ambient temperature of the human body is 37 °C, which means
many of the disease-causing bacteria are mesophiles. Mycobacterium
tuberculosis is the bacterium that causes tuberculosis in humans. It divides
every 15 to 20 hours, which is very slow when compared to other pathogenic
bacteria such as Escherichia coli, which can divide every 20 minutes.
• On the other end of the spectrum are the extremophiles. These bacteria can
survive extremely harsh conditions such as high temperatures, high salinity,
highly acidic environments and more. For instance, the Deinococcus
radiodurans is an extremophilic bacteria that can survive a thousand times more
radiation than a person can.
• Under normal circumstances, it can divide every 48 hours. However, when
exposed to harsh conditions like drought, it can slow down its growth rate until
more favorable conditions arise.

The steps involved in the binary fission in bacteria are:

Step 1- Replication of DNA
The bacterium uncoils and replicates its chromosome, essentially doubling its content.
Step 2- Growth of a Cell

After copying the chromosome, the bacterium starts to grow larger in preparation for
binary fissions. It is followed by an increase in cytoplasmic content. Another
prominent trait of this stage is that the two strands migrate to opposite poles of the
cell.
Figure 14: Binary Fission
Step 3-Segregation of DNA

The cell elongates with a septum forming at the middle. The two chromosomes are
also separated in this phase.

Step 4- Splitting of Cells

A new cell wall is formed at this phase, and the cell splits at the centre, dividing the
parent cell into two new daughter cells. Each of the daughter cells contains a copy of
the nuclear materials as necessary organelles.

TRANSFORMATION

In transformation, a bacterium takes in DNA from its environment, often DNA that's
been shed by other bacteria. In a laboratory, the DNA may be introduced by scientists
(see biotechnology article). If the DNA is in the form of a circular DNA called a
plasmid, it can be copied in the receiving cell and passed on to its descendants.

Definition

• Transferring free DNA liberated from the donor (bacteria) to the extracellular
surroundings resulting in the assimilation and typically the expression of the
hence acquired characteristics in the recipient bacteria. This phenomenon is
bacterial transformation.

• Bacterial transformation does not necessitate a living donor. The only

requirement is the free DNA. Such recipients which propagate the newly found
DNA successfully are referred to as the transformant. In harsh weather
conditions, a few genes of bacteria impulsively give out DNA from the cells to
the environment that is ready to accept through the competent cells. These cells
 in turn are responsive to modifications in the surroundings thereby regulating
the degree of gene acquisition via a natural transformation phenomenon.
• Some of the species of bacteria are evolved to pick up specific mechanisms such
as competence to uptake and hence, carry out recombination of external DNA,
which in cases involves the degradation of one strand of the incoming DNA and
incorporation of the other strand into the chromosome in a kind of homologous
recombination.

Bacterial Transformation – Basis

• The basis of bacterial transformation is the natural tendency of the bacteria to

liberate DNA that is further consumed by other competent bacteria whose
success is dependent on the competence of such host cells. Those entities which
are transformable (naturally) immediately release its DNA in the stationary
phase through autolysis.

• Some bacteria such as E.coli are treated artificially in labs so as to raise its
potential to transform through the usage of chemicals, or through the application
of a strong electric field or through the usage of a heat shock.

• Electroporation causes an increase in the competence through an increase in the

cell wall’s permeability allowing the donor DNA to enter. Likewise, the
transformants are chosen if the transformed DNA contains a selectable marker
namely the antimicrobial resistance.

• In other cases, the DNA encodes to utilize the growth factor for instance amino
acid.
Figure 15: Principal steps in bacterial transformation.
Bacterial Transformation – Mechanism

One of the key processes in molecular cloning is bacterial transformation. The final
outcome of this process is production of multiple copies of a recombinant DNA
molecule. Initial steps to create recombinant plasmids are detailed in traditional
cloning fundamentals, which includes the insertion of a desired DNA sequence into a
vector backbone. Here, the introduction of the DNA into the competent bacterial strain
occurs so that the bacteria replicate the desired sequence in quantities appropriate for
additional analysis or manipulation.

Key steps in the process of bacterial transformation are:

• Readying the competent cells

• Process of transformation

• Period for recovery of cells

• Cell plating process

Figure 16: Transformation

TRANSDUCTION:

• Transduction is a mode of genetic transfer from one bacteria to another through

a virus. There is no direct contact between the bacterial cells. The other ways of
genetic recombination in bacteria include transformation and conjugation.

• In this process, bacteriophages, which infect bacteria, use host cells to

multiplicate and while assembling they sometimes pack the bacterial DNA with
them. Later, when these viruses infect new bacterial cells, the bacterial genome
that they carry may get inserted into the host genome.

• Transduction is commonly used in genetic engineering for inserting foreign

DNA into the host cell.

• Transduction was discovered by Zinder and Lederberg in Salmonella. Hershey

and Chase used transduction as a tool to confirm that DNA is the genetic
material.

Bacterial Transduction Steps

In transduction, the transfer of bacterial DNA depends on viral infection. The steps
involve:

1. Infection of the bacterial cell by bacteriophage.

2. The virus uses the host machinery to make multiple copies either directly by the
lytic cycle or first gets incorporated into the bacterial genome by the lysogenic
cycle and followed by the lytic stage.

3. During assembly of bacteriophages, the bacterial genome also gets packed by

mistake in the viral head alongside the viral genome. In the lysogenic cycle,
 during excision of prophage, some parts of the bacterial genome that flank the
prophage are also excised and go inside the assembled viral head together with
the viral genome.

4. When these viruses infect another bacterial cell, they inject the viral DNA as
well as donor DNA into the host cell.

Figure 17: Transduction

Types of Transduction:

Transduction is common in both virulent and temperate phages, i.e. by lytic or

lysogenic cycle. Transduction is of two types:
• Generalized Transduction – In this, the phage can carry any part of DNA.
• Specialized Transduction – In this, the phage carries only the specific part of
DNA.
Generalized Transduction
• Generalized transduction can occur by both lytic or lysogenic cycle. Here, any
random part of DNA gets packed in bacteriophages by mistake along with the
viral genome. It occurs at the lytic stage of the phage life cycle.
• When the virus-containing bacterial DNA infects another cell, it can get inserted
into the host genome or if it was a plasmid, then it can reform the plasmid.
• Generalized transduction is used to study linkage information, gene mapping,
comparing genomes of two different bacteria, mutagenesis, etc.
• Example of generalized transduction includes E.coli transduction by P1 phage.

Specialized Transduction

• Specialized transduction can occur only through the lysogenic cycle, i.e. by
temperate phage. Here, only the specific part of the bacterial DNA is packed
into the virus.
• It occurs when the prophage, i.e. viral DNA, which gets inserted into the
bacterial genome in the lysogenic cycle excises.
Figure 18: Transduction types
 • When prophage excises from bacterial DNA, some parts of bacterial DNA,
which are flanked on both sides of the prophage are also excised. Here, the
newly packed phage genome consists of both bacterial and viral genome.
• Later, when the virus with the recombinant genome infects a new bacterial cell,
the bacterial gene also gets inserted into the host genome with the viral genome
through lysogeny. The recipient cell now shows the newly acquired
characteristics.
• Specialized transduction is commonly used for isolation and insertion of genes
of choice.
• Example of specialized transduction includes E.coli transduction by 𝝀 phage.
Application of Transduction
Transduction is one of the most important tools for genetic engineering.
• Transduction is used to insert the genes of choices in animals and plant cells to
modify the genetic constituents and get the desired characteristics.
• It can be used for gene therapy. It has huge potential to cure genetic diseases.
• It is an important tool in genetics and molecular biology research.

BACTERIAL CONJUGATION

• It is the transfer of genetic material between bacterial cells by direct cell-to-cell

contact or by a bridge-like connection between two cells. This takes place
through a pilus. It is a parasexual mode of reproduction in bacteria.
• It is a mechanism of horizontal gene transfer as
are transformation and transduction although these two other mechanisms do
not involve cell-to-cell contact
• Direct contact between the donor and recipient bacteria leads to the
establishment of a cytoplasmic bridge between them and transfer of part or all
 of the donor genome to the recipient. Donor ability is determined by specific
conjugative plasmids called fertility plasmids or sex plasmids.
• The F plasmid (F factor) of E. coli is the prototype for fertility plasmids in
Gram-negative bacteria.
• Strains of E. coli with an extrachromosomal F plasmid are called F+ and
function as donors, whereas strains that lack the F plasmid are F– and behave as
recipients. The conjugative functions of the F plasmid are specified by a cluster
of at least 25 transfer (tra) genes which determine expression of F pili, synthesis
and transfer of DNA during mating and other functions.
• Each F+ bacterium has 1 to 3 F pili that bind to a specific outer membrane
protein (ompA) on recipient bacteria to initiate mating. An intercellular
cytoplasmic bridge is formed, and one strand of the F plasmid DNA is
transferred from donor to recipient, beginning at a unique origin and progressing
in the 5′ to 3′ direction.
• The transferred strand is converted to circular double-stranded F plasmid DNA
in the recipient bacterium, and a new strand is synthesized in the donor to
replace the transferred strand.
• Both of the exconjugant bacteria are F+, and the F plasmid can therefore spread
by infection among genetically compatible populations of bacteria. In addition
to the role of the F pili in conjugation, they also function as receptors for donor-
specific (male-specific) phages.
 Figure 19: Conjugation

1. Donor cell produces pilus.

2. Pilus attaches to recipient cell and brings the two cells together.
3. The mobile plasmid is nicked and a single strand of DNA is then transferred to
the recipient cell.
4. Both cells synthesize a complementary strand to produce a double stranded
circular plasmid and also reproduce pili; both cells are now viable donor for the
F-factor
 • F-plasmid is an episome (a plasmid that can integrate itself into the
bacterial chromosome by homologous recombination) with a length of about
100 kb. It carries its own origin of replication, the oriV, and an origin of
transfer, or oriT. There can only be one copy of the F-plasmid in a given
bacterium, either free or integrated, and bacteria that possess a copy are
called F-positive or F-plus (denoted F+). Cells that lack F plasmids are called F-
negative or F-minus (F−) and as such can function as recipient cells.

Lesson 5: MUTATION:

• Genomes of bacteria exist on a single double-stranded circular DNA molecule

that contains approximately 4000 kb of DNA and are regulated by operons.
• A mutation is a change in the nucleotide sequence and can create new cellular
functionalities or lead to the dysfunction of others.
• Mutations can occur spontaneously or be caused by exposure to mutation-
inducing agents.

1. Gene mutation where the allele of a gene changes.

2.Chromosome mutation where segments of chromosomes, whole

chromosomes, or entire sets of chromosomes change.

Causes and Mechanisms of Mutation

• Errors in DNA replication.

• Errors in DNA repair.
• Environmental mutagen causes DNA damage that is not repaired correctly.
 • Transposons and insertion sequences (a mobile DNA elements that can move
from one location in the chromosome to another; the element may “jump” into a
gene thereby mutating it).
• External Causes: Mutagenic agents that damage DNA such as chemical
mutagens, physical mutagens or biological mutagens.

A mutation is a permanent alteration in the sequence of nitrogenous bases of a DNA

molecule. The result of a mutation is generally a change in the end‐product specified
by that gene. In some cases, a mutation can be beneficial if a new metabolic activity
arises in a microorganism, or it can be detrimental if a metabolic activity is lost.

Types of mutations:

The most common type of mutation involves a single base pair in the DNA molecule
and is known as a point mutation. In this case, a different base is substituted for the
normal base, thus altering the genetic code. Should a new amino acid be substituted in
the final protein, the mutation is known as missense mutation. Certain mutations
change the genetic code and destroy the information it contains. Such a mutation is
referred to as a nonsense mutation.

In another type of cell mutation, a frameshift mutation, pairs of nucleotides are either
added to or deleted from the DNA molecule, with the result that the “reading frame” is
shifted. The amino acid sequence in the resulting protein changes as a result of this
frameshift. If a mutation occurs without laboratory intervention, it is a spontaneous
mutation; if it occurs as a result of laboratory intervention, it is an induced mutation.

• Spontaneous mutations are the result of errors during DNA replication. When
DNA Polymerase is synthesizing a new strand of DNA, occasionally, a
nucleotide will be mispaired, added, or omitted.
• Thus, a point mutation will occur. For example, when nucleotides are mispaired,
it will appear that one nucleotide substitutes for another leading to one mutated
DNA strand. Two separate malfunctions must happen in the bacteria's DNA
replication machinery for this to occur:
• DNA polymerase pairs an incorrect complementary nucleotide base onto the
parent strand in the replication fork
• The chemical activity of the mispairing is not enough to slow the polymerase
portion of DNA polymerase so that the exonuclease can remove the mispair
• DNA bases can exist in many different forms, referred to as tautomers.
Nucleotide bases dominantly exist in the keto (C-O) and amino (C-NH2) forms,
while the imino (C≡NH) and enol (C-OH) occur rarely.
• Tautomerization, during DNA replication, will alter nucleotide base pair
formation. For example, assume that thymine undergoes keto-enol
tautomerization during replication.
• This enol species will preferentially bind to guanine during the first replication
cycle. Due to the semiconservative nature of DNA replication, at the end of the
2nd round of replication, there will be (3) A-T base pairs and (1) G-C in the
locus of mutation.
• The mechanism is as follows:
T – A --> Tautomerization --> T' – A --> replication 1 --> T' – G and A –T
T – G --> Replication 2 --> T – A and G – C
(enol form of thymine indicated as T')

• Errors in DNA replication can result in the addition of erroneous nucleotides or

the deletion of template nucleotides.
• For example, loci with a high number of short repeat nucleotides are prone to
polymerase slippage. During replication, the DNA Polymerase temporarily
 dissociates from the template strand and may relocate a few repeats upstream or
downstream of its original locus.
• Slip strand mispairing can result in addition/deletion mutations because some
nucleotides are replicated twice while others do not replicate.
• If the repeats are not in a multiple of three, the mutation can result in a
frameshift (A shift in the coding sequence downstream of the mutation).
• These mutations lead to loss of normal protein functionality.
• When an addition or deletion occurs, the potential genomic outcomes are as
follows:
• Silent mutation: The mutation changes the original codon into another codon
that codes for the same amino acid
• Missense mutation: When a mutation in the sequence causes a codon to code for
a different amino acid
• Nonsense mutation: A mutant stop codon replaces a wild-type codon, which
terminates translation resulting in a shortened protein.
• The mutation's phenotypical severity depends on the structure of the substituted
amino acid's effect on the final protein product.

Induced Mutation:
Mutagens may be of physical, chemical, or biological origin. Mostly they act on the
DNA directly, causing damage, which may result in errors during replication.

• Although severely damaged DNA can prevent replication and cause cell death.
SOS is an example of cellular response to DNA damage that results in cell cycle
arrest and induction of mutagenesis. Rec A induces SOS response by
recognizing single-stranded DNA and activating mutagenic DNA polymerases
(II, IV, and V).
 • Chemical Mutagens are agents that either directly or indirectly induce
mutations. A chemical mutagen can either replace a base in DNA, alter a base's
composition and pairing behaviour, or damage the base so that it can no longer
pair.
These include DNA reactive chemicals such as those listed below:
Base analogs:
• Structurally similar enough to nucleotides in that they can incorporate into
DNA. For example, 5-bromouracil, an analog of thymine, acts as a substrate
during DNA replication and causes point mutations. This mispairing occurs
because the base analog forms a tautomer and pairs with guanine instead of
adenine.
Reactive oxygen species:
• Hydroxyl radicals attack guanine, thereby producing 8-hydroxy-
deoxyguanosine (8-OhdG), which mispairs with adenine instead of cytosine,
which resulting in a (G -> T) transversion during replication.
Deaminating agents:
• These agents remove amino groups on nucleotide bases. Deaminating agents
produce an adenine species that pairs with cytosine and a
cytosine species (Uracil) that pairs to adenine. Deamination of guanine results in
xanthine, which inhibits replication, thereby not creating a mutation.
Flat aromatic compounds:

• Acridines like ethidium bromide can intercalate with adjacent pyrimidine base
pairs. This interaction slightly unwinds the helix and increases the distance
between adjacent base pairs. This intercalation disrupts the reading frame during
translation and can cause insertions or deletions.
Figure 20: Mutation types
Alkylating agents:

• Agents like ethyl methanesulfonate and dimethyl nitrosoguanidine alter the

nucleotide base by adding alkyl groups. The nature and position of the
alkylation can vary but usually leads to point mutations through base
mispairing. However, alkylation can cause crosslink formation, which
inhibits replication.

Ames test:

Figure 21: AMES test