Microbiology Basic and Applied

Dr. Bipinraj N K

Microbiology
Microbiology is the study of microorganisms.
Microorganisms are all single-celled microscopic
organisms and include the viruses, which are microscopic
but not cellular.
Nutrition
Movements
Growth
Reproduction
React to the environment
Produce various extracellular compounds

Role of Microbiology

Medical
Industrial
Molecular Biology
Environmental
Genetics & Recombinant DNA Tech

Microorganisms or Microbes
Microscopic organism
Bacteria
Fungi
Algae
Protozoa
Virus

General Characteristics of Bacteria

A prokaryotic microorganism(no membrane-enclosed nucleus)

Size: average size 0.5 m
Shape :
No mitochondria or chloroplasts: How it generate energy?
Single chromosome. There are reports of more than one in few
microorganisms e.g Rhodobacter sphaeroides, Vibrio cholerae
A closed circle of double-stranded DNA (Plasmid): Is there any
application of plasmid?
Flagella may present (made up of protein flagellin): Is there any
industrial advantage of having flagella?
Ribosome present
Rigid cell wall made of peptidoglycan. (Gram + and -)
The plasma membrane : phospholipid bilayers
Reproduction : Asexual by fission or spore formation
Sexual by conjugation

 Produce industrially important molecules

Eg ?
Various mode of nutrition : any advantage?
Aerobic and anaerobic catabolism : any advantage?
Energy storage mechanism in the form of polymers. Eg?
Production various virulence factor. What is a virulence factor
and its applications?
Diverse habitat, ability to grow in extreme conditions. any
application?

Bacterial Cell Wall :G +

Teichoic acids, polymers of glyce

or ribitol joined by phosphate gro

It has negative charge

80 nm

60 nm

Bacterial Cell Wall : G -

8 nm

Eukaryotic organisms
Large, diverse, and widespread group of organisms, consisting of the molds,
mushrooms, and yeasts.
Approximately 100,000 species of fungi have been described, and as many as
1.5 million species may exist.
Most closely related to animals
Most fungi are terrestrial. They inhabit soil or dead plant matter and play
crucial roles in the mineralization of organic carbon.
A large number of fungi are parasites, cause many of the economically
signicant diseases of crop plants and in animals, including humans (mycosis)
Fungi also establish symbiotic associations with many plants, facilitating the
plants acquisition of minerals from soil, this association is called as
mycorrhizae. (ectomycorrhizae, eg. basidiomycetes and endomycorrhiza eg.
glomeromycete )
Many fungi benet humans through fermentation and the synthesis of
antibiotics.

Fungus
Fungi are chemoorganotrophs
Fungi feed by secreting extra- cellular enzymes that
digest complex organic materials, such as
polysaccharides or proteins, into sugars, peptides,
amino acids, and so on, which are assimilated as
sources of carbon and energy.
Many fungi can grow at extremes of low pH or high
temperature

 Cell structure
Single cellular: yeast
Multicellular : mold
Long, branched, threadlike laments of cells
called hyphae
Septate
Ceonocytic

Hyphae typically grow together across a

surface and form a compact,
macroscopically visible tuft called a
mycelium
From the mycelium hyphal branches reach
up into the air and form asexual spores
called as conidia, mostly pigmented.
Some fungi form macroscopic
reproductive structures called fruiting
bodies

 Most fungal cell walls consist of chitin, a

polymer of the glucose derivative Nacetylglucosamine.
Chitin is arranged in the walls in
microbrillar bundles to form a thick, tough
wall structure.
Other polysaccharides such as mannans
and galactosans are present on the cell wall
Fungal cell walls are typically made up of
8090% polysaccharide, along with
proteins, lipids, polyphosphates, and
inorganic ions making up the wallcementing matrix.

What are the applications of such a complex structure?

 Dimorphism
Dimorphic fungi can change from the yeast (Y) form in the
animal to the mold or mycelial form (M) in the external
environment
Various factors controls this YM shift (nutrients, CO2,
oxidation-reduction potentials, temperature).
In plants the M form occurs in the plant and the Y form in
the external environment.

 Reproduction
Asexual : Fission, Budding, asexual spore

 Sexual reproduction
Some fungi produce spores as a result of sexual
reproduction. The spores develop from the fusion of either
unicellular gametes or specialized hyphae called
gametangia
Sexual spores can also originate from the fusion of two
haploid cells to yield a diploid cell; this then undergoes
meiosis and mitosis to yield individual haploid spores.
Homothalic : self-fertilizing and produce sexually compatible
gametes on the same mycelium
Heterothalic: out-crossing between different but sexually
compatible mycelia
If Spores formed within an enclosed sac (ascus) are called
ascospores,
Spores produced on the ends of a club-shaped structure
(basidium) are basidiospores
Zygospores, produced by zygomycetous fungi, such as the
common bread mold Rhizopus are macroscopically visible
structures that result from the fusion of hyphae and genetic
exchange.

Phylogeny
Chytridiomycetes
Single cell or mycelial, Produces zoospores

Zygomycetes
Coenocytic, commonly found in soil and on
decaying plant material.
Produce zygospores

Glomeromycetes
Symbiotic relationships with plant roots
Produce zygospores

Ascomycetes
Single celled, eg Yeast

Basidiomycetes
Eg. Mashroom

Role of fungi in Biotechnology

Drugs (antibiotics)
Food
Pesticides
Pollution control
Study organism (S cerevisiae, Neurospora crassa )

Nanoarchaeota

Archaea
Archaea are quite diverse group
Gram positive or gram negative some lack
cell wall
Shape spherical, rod-shaped, spiral, lobed,
plate-shaped, irregularly shaped, or
pleomorphic.
Some are single cells, whereas others form
laments or aggregates.
Size range from 0.1 to over 15m
Multiplication ; by binary fission, budding,
fragmentation,

Archaea

(a) Heliobacterium modesticaldum; the cell measures 1x3 m. (b) Methanopyrus kandleri; the cell measures 0.5 x 4
m. (c) Saccharomyces cerevisiae; the cell measures 8 m in diameter.

 Respiration: aerobic, facultatively, anaerobic, or

strictly anaerobic.
They can be autotrophic, chemoorganotrophic
or chemolithotrophic
Some are mesophiles; others are hyperthermophiles
that can grow above 100C.
Some are extreme halophile, some are extreme
acidophiles

 Cell structure: Most cases it is made up of

pseudomuramic acid (alternating groups of Nacetylglucosamine and N-acetyl talosamin uronic
acid with (14) glycosidic bonds
Some cases pseudomurin is replaced by thick
polysaccharide walls composed of polymers of
glucose, glucuronic acid, galactosamine uronic
acid, and acetate. As in the case of extreme
halophilic archaea. They also contains SO 42- which
reacts with high Na+ concentration in the habitat.
Some archaea cell wall is made up of Surface
layer or S-layer consists of an interlocking
protein or glycoprotein molecules arranged to
form various symmetries such as hexagonal or
tetragonal

 Cell membrane: Mainly three types

Lipid bilayer of glycerol diethers
Monolayer of Glycerol tetra ether
Monolayer of biphytanyl tetraether
Mixture of both monolayers are also seen

Bilayer of C20diethers

Monolayer of C tetraethers.

 This lipid monolayers are resistant to heat

Different cyclic rings are also present in the lipids of
archaea imparting various properties to the
membrane

Phylogeny
Classication:
The rst edition of Bergeys Manual divided the archaea into ve major groups based on physiological and
morphological differences

Methanogenic archaea,
Archaea sulfate reducers,
Extremely halophilic archaea ,
Cell wallless archaea ,
Extremely thermophilic S0 -metabolizers

On the basis of rRNA data archaea are divided into four

Euryarchaeota: physiologically diverse group. Most of them are extreme
acidophiles or exreme halophiles
(7 classes : Methanobacteria, Methanococci, Halobacteria, Thermoplasmata, Thermococci,
Archaeglobi, and Methanopyri)

Crenarchaeota: Mostly hyper thermophilic group

Korarhcaeota: not yet recognized as a separate group, mostly similar to the
primitive archaea
Nanoarchaeota : A parasitic prokaryote usually seen attached to Ignicoccus a
Crenarchaeota

Survival strategies
Extremely Halophilic Archaea: requires more that 1.5
M (about 9%) NaCl for growth
Pump large amounts of K+ from the environment into the
cytoplasm
High content of acidic aminoacids aspartate and
glutamate in the glycoproteins of the cell wall,
Acidic proteins in cytoplasm , ribosomes etc.
Presence of bacteriorhodopsin to synthesis ATP under
anoxic conditions

 Reference : Chapter 3: Cell structure and function of

bacteria and Archaea

Nanoarchaeota

Cyanobacteria
What is the importance
of cyanobacteria in the
evolution of life on
Earth?

Cyanobacteria (Blue green algae)

Morphologically diverse large group

of oxygenic photosynthetic bacteria
Shows gliding movement
(hydrotaxis)
size 0.5 - 40 m dia
GC ratio 35 70% variation

 Structure:
Peptidoglycan cell wall
Many produce excessive mucilaginous envelops
Multilayered photosynthetic layers with two types of pigments
: Phycobilins and Chll a
Photosystem I and II are present for using atm CO2
Gas vesicles
Some forms heterocyst with repeated cluster of nif genes
Nitrogen storage structure, cyanophycin (asp and arg)

 Nitrogen xation:
Nitrogen xation is performed in a specialized structure called as
heterocyst
It contains nitrogenase enzyme
It has three additional cell walls which contains one layer of
glycolipids
It lacks photosystem II
Polar plugs contains cyanophycin that reduces cell to cell diffusion
Fixed nitrogen (glutamine) are diffused from other cells to
heterocyst

 Different groups:
Unicellular divide by binary ssion (Unicellular)
Unicellular divide by multiple ssion forms colony
(Plurocapsalean)
Non-heterocystous laments (Oscillatorean)
Filamentous with differentiated cells, heterocyst
(Nostoclean)
Branching laments (Branching)

Applications

Cyanobacterial bioactive compounds

Cyanobacterial bioplastics
Cyanobacterial consortia for bioremediation purposes
Cyanobacterial alternative energy sources
Cyanobacteria as biofertilizers
Cyanobacteria as a healthy food source

Cyanobacterial emulsifiers
exopolysaccharides

 Cyanobacterial toxins
Many cyanobacteria are known to produce toxins
Eg.
Microcystins

Liver

Microcystis, Anabaena,
Planktothrix etc.

Nodularin

Liver

Nodularia

Anatoxin-a

Nerve synapse

Anabaena, Planktothrix

 Cyanobacterial mats
Cyanobacterial bloom
Symbioses
Fungi form symbiotic associations with cyanobacteria and
are called lichens.

Actinomycetes
or Ray Fungus
Gram + rod non-motile bacteria
Branching laments forms thallus and
mycelium
Aerobic as well as facultative aerobics
Predominantly present in soil responsible
for earthy smell
Spore forming (Conidial spores)
Four types of cell wall based on amino
acid or glycine in the peptidoglycan
interbridge
Important species are :
Actinomyces, Nocardia, Dermatophillus,
streptomyces ets

Applications of actinomycetes
Nitrogen xing (actinorhizae)
Antibiotic producing
Industrial enzymes
Used for bioconversions
Pathogenicity

Microbial Nutrition
Macro elements
Micro elements
Growth factors

 Requirement of C H O
Mostly Satised together
Compounds act as source for C,H and O as well as energy
source
Eg of Carbon sources
CO2, organic carbon sources

Oxygen classes of Microorganisms

Aerobes

Anaerobes

Obligate

Require O2

Micrococcus
luteus

Aero tolerant

Facultative

Not required,
but grow better
with O2

E. Coli

Obligate

Microaerophilic

Required, but at
low level

Spirillum
volutans

Obligate aerobes
Facultative aerobe
respiration
Microaerophilic

:
:
:

Aerobic respiration
Aerobic respiration, Fermentation and

Aerobic respiration

Obligate anaerobes :
Fermentation
Aerotolerant
:
Fermentation and

Anaerobic respiration

Not required, but

can tolerate O2

Streptococcus

O2 is harmful

Methanobacterium

Anaerobic

 Cultivation of aerobes and

anaerobes
Aerobes : agitation or bubbling to
provide O2
Anaerobes: various method to
stop O2
Filling the bottles completely and
close with tight cap
Addition of reducing agents to
convert O2 to H2O
Thioglycolate

Bubbling the medium with N2 or H2S

Culturing in anoxic jar or glove box

 Requirements for Nitrogen, Phosphorus and

Sulfur
Needed for the synthesis of biomolecules
Nitrogen is obtained from organic (amino acids, DNA,) or
inorganic (nitrate, nitrite,) source
Phosphorus is obtained from organic as well as inorganic
sources
Organic P is converted to inorganic from by alkaline phosphatase

Sulfate is the preferred source of sulfur.

 Growth Factor
Organic compounds required because they are essential
cell components or precursors of such components and
cannot be synthesized by the organism are called growth
factors.
Major classes of growth factors:
(i) Amino acids, (ii) Purines and pyrimidines, and (iii) vitamins.

Uptake of Nutrients
Passive Transport System
Passive diffusion
Facilitated diffusion

Active Transport system

Active transport
Group translocation

 Passive diffusion: movement of molecules from a region of

higher concentration to one of lower concentration
The rate of passive diffusion is dependent on the concentration
gradient between a cells exterior and its interior
A fairly large concentration gradient is required for adequate
nutrient uptake by passive diffusion , and the rate of uptake
decreases as more nutrient is acquired unless it is used
immediately.
Very small molecules such as H 2O, O2, and CO2 often move across
membranes by passive diffusion

 Facilitated diffusion: Passive diffusion aided by a

facilitator is called as facilitated diffusion.
The rate of facilitated diffusion increases with the
concentration
There are two types of facilitators
Carrier proteins : Embedded in the cytoplasmic membrane
and are nutrient-specic.
Eg. Aquaporin Z, Glycerol facilitator
Porin proteins : Embedded in the outer membrane and
facilitate the movement of molecules from the cell exterior to
the cell periplasmic space. They also allow molecules to flow in
the reverse direction; two types nonspecific porins (Omp F)
& substrate-specific porins (Siderophores complexes)

Siderophore
Siderophores are low molecular weight
molecules that are able to complex with ferric
iron and supply it to the cell.
E. coli produce cyclic catecholate
Eg. Enterobactin

Microorganisms produce siderophore when the

conc. Of Fe is less
Iron binds with siderophore making a complex.
This complex, which can be taken inside the
cell through siderophore binding receptor.

Active transport
Active transport characteristic features:
Transport of solute molecules even external concentration
is low.
Use of energy in the form of ATP or proton motive force
Involvement of carrier protein
Can be inhibited by metabolic inhibitors

 Eg. ATP-binding cassette transporters (ABC transporters) : comprise a

large class of active transport systems, deliver molecules across the
cytoplasmic membrane with the concomitant hydrolysis of ATP as the
driving force for molecule uptake
Three protein components make up a typical ABC transporter
a) A solute binding protein,
b) A trans-membrane spanning protein,
c) An ATP hydrolyzing protein that energizes the uptake process.

A proton gradient also can power active transport

indirectly, often through the formation of a sodium ion
gradient

Group translocation
A process in which a molecule is transported into the
cell while being chemically altered.
phosphoenolpyruvate: sugar phosphotransferase system
(PTS).
PTS is a complex system, consists of 3 proteins
1. Heat stable protein
2. Enzy I or EI
3. Enzy II, or EII, (has subunits EIIA, EIIB and EIIC).

 http://
ecolistudentportal.org/article_nutrient_transport

Bacterial Growth
Increase in the number of cells in a population
What is the need of studying bacterial growth?

Cell division

DNA replication and cell elongation

Formation of cell division plane
Synthesis of petidoglycan
Cell division

 Generation time: time required for one cell to divide

and form two cells
Binary ssion
During growth cycle all cellular constituents
increases proportionally and each daughter cell
receives chromosomes and sufficient copies of
ribosomes and other monomers.

 What triggers cell division in bacteria?

How is it controlled?
In Rod shaped bacteria how constriction happens
exactly at the center ?

 Control of Cell Division :

Fts Proteins and divisome
Min proteins (minicell locus ) (Min C, Min D and Min E)
Nucleoid occlusion mechanism

Fts Proteins
Fts (filamentous temperaturesensitive) Proteins
Essential for cell division in all
prokaryotes
Interact to form the divisome (cell
division apparatus)
FtsZ: forms ring around center of cell
ZipA: anchor that connects FtsZ ring to
cytoplasmic membrane
FtsA: helps connect FtsZ ring to
membrane and also recruits other
divisome proteins
Related to actin
FtsI peptidoglycan biosynthesis proteins
FtsK separates chromosome

 DNA replicates before the FtsZ ring forms

Location of FtsZ ring is facilitated by Min proteins
(minicell locus )
MinC, MinD : Inhibits FtsZ anchoring
MinE : Inhibitor of MinC and MinD present at the center

FtsK protein mediates separation of chromosomes to

daughter cells

1. DNA replication and segregation

2. Min system controls the site of
division
3. FtsZ ring assembly in the center
along with the formation of
divisome
4. Z-ring maturation
5. Septal invagination with the
constriction of the envelope layers
6. Septum closure and splitting of the
daughter cells

 FtsZ ring assembly

1. First group of proteins
assembles and anchor the
FtsZ to form Z ring
2. Z ring forms as an arc at
several points at the median
3. Arc formed by the self
polimerization of FtsZ
proteins with the help of GTP
utilization
4. FtsA and ZipA proteins anchor
the micro arc on the cell
membrane

 Z-ring maturation after

1. By the ordered recruitment
of late divisome components
2. ZapA and ZapC enhance the
polymerization of the arc by
aggregating the FtsZ.
3. FtsK drag Chromosome from
the plane

 Septal invagination with the constriction of the

envelope layers
Constriction is energy dependent
Z-ring can exert a constrictive force onto the membrane
Simultaneously autolysin hydrolyses peptidoglycan layer
and create small opening on the cell wall
New wall materials are added across the opening with the
help of bactoprenol and PBP

Control of Z ring assembly

By Two complementary systems ; Min system and
Nucleoid occlusion
Min system prevents aberrant division at the poles
Fts inhibitor MinC and MinD organized to form MinCD
complex
MinCD prevents the lateral assembly of FtsZ
Competes with FtsA and ZipA
Min E system prevents MinCD complex

Regulation of Z ring ASSEMBLY

 Nucleoid occlusion
DNA associated proteins inhibits Z-ring formation
This provides a protective mechanism to the DNA, and
contributes to the precise temporal and spatial positioning
of the division septum.

 Growth rate dependent Z ring inhibitor

UgtP (UDP-glucose diacylglycerol glucosyltransferase) was identied in B.
subtilis and may constitute the link between metabolism and cell
division
Under nutrient rich conditions, in response to high levels of its
substrate, UgtP is present in the cytoplasm inhibits FtsZ
assembly, by disruption of the lateral protolaments interactions
during growth on a poor carbon source, the levels of UgtP are
reduced

When DNA is damaged, an SOS response is activated to

repair the DNA and cease cell division by inhibiting FtsZ
polymerization

Bacterial growth

Microbial growth is defined as the increase

in number of cell in a population.

Growth Curve: graphical expression of

microbial growth.

Lag, log, stationary and death phase

Lag phase: When a microbial culture is

inoculated into a fresh medium, growth
usually begins only after a period of time
called the lag phase.

Log phase : phase in which the cells double

during a constant interval.

Stationary Phase: In the stationary phase,

there is no net increase or decrease in cell
number and thus the growth rate of the
population is zero.

Death Phase : phase in the growth when the

cells start dying and the graph curves
downwards

 Lag Phase: Lag phase is the result of change of

conditions for microorganisms
Duration of lag phase depends on various
parameters such as; temperature, pH, nutrients and
age of the culture
Lag phase is the time taken to adjust with the new
growing conditions.
How to reduce the duration of lag phase ?

 Log phase is the phase in which bacteria show exponential

growth.
During this phase generation doubles with constant interval
As a result the graph shows a straight line in a semilogarithmic
graph
Metabolic activity will be on high rate in log phase.
This phase is used for the calculation of generation time
Rates of exponential growth vary greatly. The rate of exponential
growth is influenced by environmental conditions (temperature,
composition of the culture medium), as well as by genetic
characteristics of the organism itself.
Generation time can be calculated by formula
G = 3.3 (log N - log N0)/ t

Stationary Phase
In a batch culture exponential growth is limited.
After exponential growth essential nutrients is almost used up and bacteria
produce large amount of waste products
This conditions reduce growth rate
Bacterial metabolism reduces
Some cells divide and some die as a result growth rate become almost zero
This is a phenomenon called cryptic growth
Usually during stationary phase bacteria produce many secondary metabolites
During this stage bacteria frequently produce a variety of starvation proteins,
which make the cell much more resistant to damage in a variety of ways
They increase peptidoglycan cross-linking and cell wall strength
As a result of these and many other mechanisms, the starved cells become
harder to kill and more resistant to starvation itself, damaging temperature
changes, oxidative and osmotic damage, and toxic chemicals such as chlorine.

 Death Phase
Detrimental environnemental changes decrease in
nutrients and the buildup of toxic wastes lead to the
decline in the number of viable cells characteristic of
the death phase.
Some time the dead cell fail to lyse as result the
optical density of the medium can remain constant
and the graph can be a straight line.

 Measurement of Microbial growth:

Direct counting
The Petroff-Hausser Counting Chamber
Plating methods
TVC
Filtration method

Measurement of cell mass

Wet weight
Dry weight

Measurement of cell density

Optical density by spectrophotometer

Pure culture
In natural habitats microorganisms usually grow in complex,
mixed populations containing several species. This presents a
problem for the microbiologist because a single type of
microorganism cannot be studied adequately in a mixed culture.
One needs a pure culture, a population of cells arising from a
single cell, to characterize an individual species.
Pure culture technique was rst developed by the German
bacteriologist Robert Koch
Methods used to purify bacteria
Spread Plate, Streak Plate and Pour Plate

Sterilization
Sterilization is a process of removing or killing of
microorganism from an object such as medium,
glassware, table surface equipment etc.
Heat : moist heat and dry heat
Filtration
Chemicals
Radiation

 Disinfection : Killing, inhibition or removal of

microorganisms that may cause disease. Usually it is
applied on inanimate object
Sanitization : removal or killing of microorganism to a
level that are considered to be safe by public health
standards.

 Heat :
Autoclaving : moist heat
Hot air oven : dry heat

 Filtration:
Filtration is a way to reduce the microbial population in
solutions of heat-sensitive material
There are two types of filters
Deapth filters
Membrane filters

 Radiation:
Ultraviolet (UV) radiation around 260 nm
Ionizing radiation such as Gamma from Cobalt 60

 Factors controlling effectiveness of antimicrobial

effectiveness
Population size
Population composition
Concentration or intensity of an antimicrobial agent
Duration of exposure
Temperature
Local environment
pH
Organic compounds present

Control of Microorganism
Sterilization:
Disinfection:
Pattern of Microbial death:
Exponential

DNA Replication

Semiconservative mechanism
Enzymes that catalyze the addition of deoxynucleotides are called
DNA polymerases
There are ve different DNA polymerases in Escherichia coli, called
DNA polymerases I, II, III, IV, and V
DNA polymerases synthesize DNA in the 5 to 3 direction
To start a new chain, a primer, a nucleic acid molecule to which DNA
polymerase can attach the rst nucleotide, is required. In most cases
this primer is a short stretch of RNA.

 Initiation
Elongation
termination

Initiation of DNA Synthesis

Formation of replication fork ; The enzyme DNA helicase
unwinds the double helix, using energy from ATP, and exposes a
short single stranded region
Helicase moves along the DNA and separates the strands just in
advance of the replication fork. The single-stranded region is
covered by single strand binding protein. This stabilizes the singlestranded DNA and prevents the double helix from re-forming
DNA gyrase travels along the DNA ahead of the replication fork
and inserts negative supercoils to cancel out the positive
supercoiling
The origin of replication (oriC) consists of a specic DNA sequence
of about 250 bases that is recognized by initiation proteins, DnaA,
which binds to this region and opens up the double helix before
helicase.
Next to assemble is the helicase (known as DnaB), which is helped
onto the DNA by the helicase loader protein (DnaC). Two helicases
are loaded, one onto each strand, facing in opposite directions.
Next, two primase and then two DNA polymerase enzymes are
loaded onto the DNA behind the helicases.
Primase adds RNA primer to begin DNA synthesis
Initiation of DNA replication then begins on the two single strands.
As replication proceeds, the replication fork appears to move along
the DNA

 Synthesis of the New DNA Strands

DNA synthesis replication always proceeds from 5 to 3
Leading strand and lagging strand
After synthesizing the RNA primer, primase is replaced by
Pol III

 Each polymerase is held on the DNA by a sliding clamp, which encircles and
slides along the single template strands of DNA.
Consequently, the replication fork contains two polymerase core enzymes and
two sliding clamps, one set for each strand.
However, there is only a single clamp-loader complex. This is needed to assemble
the sliding clamps onto the DNA. After assembly on the lagging strand, the
elongation component of Pol III, DnaE, then adds deoxyribonucleotides until it
reaches previously synthesized DNA At this point, Pol III stops.
The next enzyme to take part, Pol I, it removes the RNA primer
When the primer has been removed and replaced with DNA, Pol I is released. The
last phosphodiester bond is made by an enzyme called DNA ligase which seals
the nicks in DNA

 Termination:
InE.coli, there are 10 replication termini (Ter) located in a
region opposite to the replication origin
The Ter sites interact with the replication terminator
protein called Tus, to stops DNA unwinding activity of DnaB
At the end, replication forms as catenated (two circular
chromosomes joined at ter region) ring.
Catenated rings are separated by topoisomerases IV
Topoisomerase IV transiently breaks both DNA strands of
one chromosome and allowing the other chromosome to
pass through the break

Plasmids

Plasmid Replication
Two methods
Theta formation
Rolling circle replication

Rolling circle replication

Ori

RepA

3 OH

RepA: Binds at the Ori, forms a nick on one

strand and remain attached to the 5 end of the
strand
Free 3 end with free OH group act as the
primer for Bacterial DNA polymerase III to start
replication of plasmid.
DNA PolII

Helicase

Helicase unwind the double strand, simultaneously

single strand binding protein binds to the strand in
which Rep A is attached and stabilize the strand and
progressively peeled off from the plasmid.
Once the replication of the intact strand is complete
the RepA joins the two ends of the nicked strand.

Ss binding
protein

DNA ligase seals the nick of the double stranded DNA

Now the peeled single stranded DNA forms loop like
structure allowing RNA polymerase to bind on it and
prime the replication.
DNA polymerase starts the replication and forms
dsDNA

RNA poly
Ligase

Mobile DNA: Transposable Elements

Mobile DNA refers to discrete segments of DNA that move as units from one
location to another within other DNA molecules
They are always found inserted into another DNA molecule such as a plasmid, a
chromosome, or a viral genome.
Transposable elements do not possess their own origin of replication. Instead, they
are replicated when the host DNA molecule into which they are inserted is
replicated.
Transposable elements move by a process called transposition that is important
both in evolution and in genetic analysis. (1 in 1000 to 1 in 10,000,000 per
generation)
The two major types of transposable elements in Bacteria are insertion sequences
(IS) and transposons.
Both elements have two important features in common:
They carry genes encoding transposase, the enzyme necessary for transposition, and they
have short inverted terminal repeats at their ends that are also needed for transposition.

 Insertion sequences are the simplest type of

transposable element. They are short DNA segments,
about 1000 nucleotides long, and typically contain
inverted repeats of 1050 bp.
Each different IS has a specic number of base pairs in
its terminal repeats. The only gene they possess is for
the transposase.
Several hundred distinct IS elements have been
characterized.

IS2 is an insertion sequence of 1327 bp with inverted

repeats of 41 bp at its ends.

 Transposons are larger than IS elements, but have the same two essential
components: inverted repeats at both ends and a gene that encodes
transposase.
The transposase recognizes the inverted repeats and moves the segment
of DNA flanked by them from one site to another. Consequently, any DNA
that lies between the two inverted repeats is moved and is, in effect, part
of the transposon.
Some transposons (Conjugative Tn ) are capable of moving to another
bacteria through conjugation
Composite Tn : As in the case of Mu virus the genome itself act as an Tn.
The whole structure can move as single unit.

 Signicance of transposition:
Evolution
Mutation

Pure culture concepts

Pure culture?
How to obtain pure cultures?

 Fts proteins : group of proteins involved in cell

division
Divisome: division apparatus formed by Fts proteins.
Involved in peptidoglycan sysnthesis
Synthesis of new cytoplasmic membrane

 Peptidoglycan synthesis
Autolysin makes cut in the existing peptidogylcan
Bactoprenol transport peptidogycan precursors through
cytoplasmic membrane to periplasm
Transpeptidation : Controlled by FtsI

Microscopy

 Optical phenomenon used in microscopy are:

Reflection (change in direction of awavefrontat aninterfacebetween
two differentmediaso that the wavefront returns into the medium
from which it originated)
Diffraction (Change in the directions and intensities of a group of
waves after passing by an obstacle or through an aperture whose size
is approximately the same as the wavelength of the waves)
Refraction (change in direction of propagation of awavedue to a
change in itstransmission medium)

Bright eld, dark eld, phase contrast, fluorescence

microscope.

Resolution is the ability of a lens to separate or

distinguish between small objects that are close
together.
The minimum distance (d) between two objects
that reveals them as separate entities is given by
the Abbe equation, in which lambda () is the
wavelength of light used to illuminate the specimen
and n sin is the numerical aperture (NA).
Max value of sin is 1 (90o )

Resolution:

 Magnication : Product of the magnication of its

objective and ocular lenses
Resolution is a function of the wavelength of light
used and a characteristic of the objective lens known
as its numerical aperture, a measure of lightgathering ability.
Lenses with higher magnication typically have
higher numerical apertures

Maximum theoretical resolving power of a microscope with oil immersion is

Dark Field Microscopy

Observing live objects

unstained cells and organisms can be observed by simply changing the way in which they are
illuminated.
A hollow cone of light is focused on the specimen in such a way that unreflected and unrefracted rays do
not enter the objective. Only light that has been reflected or refracted by the specimen forms an image

Phase Contrast Microscopy

A phase-contrast microscope converts slight
differences in refractive index and cell density into
easily detected variations in light intensity
Phase-contrast microscopy is used for
Studying microbial motility
Determining the shape of living cells
Detecting bacterial components

 Atomic Force Microscopy

In atomic force microscopy, a tiny stylus or a probe is
positioned extremely close to the specimen such that weak
repulsive forces are established between the probe on the
stylus and atoms on the surface of the specimen.
During scanning, the stylus surveys the specimen surface,
continually recording any deviations from a flat surface. The
pattern that is generated is processed by a series of
detectors that feed the digital information into a computer,
which then outputs an image

 Differential Interference Contrast Microscopy

Confocal Microscopy
Electron Microscopy

 The illumination is achieved by

scanning one or more focused
beams of light, usually from a
laser, across the specimen
The images produced by
scanning the specimen in this
way are called optical
sections, it enables to obtain
3D image of the specimen.

 Differential interference contrast (DIC) microscopy is a form of light microscopy that employs a polarizer in
the condenser to produce polarized light (light in a single plane).
Its an excellent method for observing transparent specimen.
The polarized light then passes through a prism that generates two distinct beams. These beams traverse
the specimen and enter the objective lens where they are recombined into one.
Because the two beams pass through different substances with slightly different refractive indices, the
combined beams are not totally in phase but instead create an interference effect.
This effect visibly enhances subtle differences in cell structure. Thus, by DIC microscopy, cellular structures
such as the nucleus of eukaryotic cells or endospores, vacuoles, and granules of bacterial cells, appear
more three-dimensional.
DIC microscopy is typically used for observing unstained cells because it can reveal internal cell structures
that are nearly invisible by the bright-eld technique.

Gene Transfer in Bacteria

Transformation: Uptake of free DNA
Transduction: DNA transfer through a Virus
Conjugation : Direct DNA transfer from one bacteria
to other

 Transformation, discovered by Fred Griffith in 1928.

Transformation is the uptake by a cell of a naked DNA
molecule or fragment from the medium and the
incorporation of this molecule into the recipient
chromosome in a heritable form.
In natural transformation the DNA comes from a
donor bacterium.
The process is random, and any portion of a genome
may be transferred between bacteria.

 Competence:
A cell that is able to take up DNA
and be transformed is called as a
competent cell.
Competent requires:
Membrane bound DNA binding
proteins
Membrane bound Nucleases
Competent specic proteins

Transduction
Transduction is the transfer of bacterial genes
by viruses.
Generalized Transduction
Specialized Transduction

 Generalized Transduction: occurs during the lytic

cycle of virulent and temperate phages and can
transfer any part of the bacterial genome.
During the assembly stage, when the viral
chromosomes are packaged into protein capsids,
random fragments of the partially degraded bacterial
chromosome also may be packaged by mistake.
The resulting virus particle often injects the DNA into
another bacterial cell but does not initiate a lytic cycle

Specialized Transduction
In specialized or restricted transduction, the
transducing particle carries only specic portions
of the bacterial genome
Specialized transduction is due to an error in the
lysogenic life cycle.

Conjugation
Bacterial conjugation (mating) is a
mechanism of genetic transfer that
involves cell-to-cell contact.
Conjugation is a plasmid encoded
mechanism. Conjugative plasmids
use this mechanism to transfer copies
of themselves to new host cells.
Conjugation involves a donor with a
conjugative plasmid (F plasmid) and a
recipient.

Mechanism of DNA Transfer During Conjugation

Hfr Strains

Mutation
A mutation is a heritable change in the base
sequence of the nucleic acid in the genome of an
organism or a virus or any other genetic entity.
Wildtype

Spontaneous mutation
Induced mutation
Point mutation
Addition
Delition
Back mutation
Reverse mutation

Molecular Basis of Mutation

Base-Pair
Substitutions

 Fame shift
mutation

 Mutagenesis
Chemical Mutagens
Base analogue

Physical
Radiation (UV)
Ionizing radiation (X Ray , Gama rays, etc)

DNA Repair Systems

Direct reversal :
By removal of the altered group
Photoreactivation

Repair of single-strand damage

Damage is removed from one strand
Base excision repair
Nucleotide excision repair and mismatch repair

Repair of double-strand damage

By recombinational mechanisms

Ames Test
The Ames test makes practical use of bacterial
mutations to detect potentially hazardous chemicals
in the environment
Bacterial tests for carcinogen screening were
developed primarily by Bruce Ames and colleagues
at the University of California in Berkeley and
consequently, the mutagenicity test for carcinogens
is known as the Ames test