MICROBIAL GENETICS

• The principles of Genetics applied to bacteria and viruses

• Genetic principles are universal
• Advances in Genetic process lead to fundamental advances
in Biology and Biochemistry
• Bacteria became the experimental tools for unraveling the
basic knowledge of genetics at molecular level

Advantages of bacteria for genetic experiment

▪Bacterial cultures contains millions of cells

▪Prokaryotic cell contains a single chromosome

(immediate, observable change in characteristics)

▪Rapid growth rate

▪Ease of growing in controlled environment

▪Great divesrsity of metabolic types among

microorganisms

 Structure of DNA

• Attached to each
Deoxyribose and
phosphate residues
arranged alternatively
• Attached to each
Deoxyribose are of four
nitrogen bases
• Purines - Adenine,
Guanine
• Pyramidine
Thymidine and Cytosine

How RNA differs from DNA

• RNA contains - Sugar Ribose instead of

Deoxyribose
• Uracil is present instead of Thymine
• Types of RNA

Messenger RNA mRNA

Ribosomal RNA r R N A
Transfer RNA tRNA
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 Knowledge on DNA lead to advances in

Molecular Biology
• Central dogma of Life – Deoxyribonucleic acid
• DNA carries the Genetic information
• DNA is transcribed to RNA – Polypeptides Cell
Function depends upon specific polypeptides –
Proteins – Enzymes
DNA is a store house of Protein synthesis DNA acts a
Template for synthesis of mRNA
•Virus differs from other as they contains either
DNA or RNA

What is a Code in Genetics

• Code is a unit consists of sequence of three Bases

• Code is triplet A-T- C
• A code can make single Amino acid
• More than one code present for making similar sequence of
Amino acid
• AGA make Arginine
• AGC, CGU, CGG, also code for similar Amino acid
• Some Codons UAA don't code for any Amino acid called as
Nonsense codon

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 How bacterial Genome differs from

Higher forms of Life

• Several stretches of DNA don't appear to function

as codons, occurs between the coding sequences of
Gene. called as INTRONS.
• Coded are called as EXONS
• In transcription introns are excised when form
RNA before translated by ribosomal proteins
• Contains a Double stranded molecules of DNA
arranged in circular form

Extra chromosomal Genetic Elements

▪ Bacteria posses Extra chromosomal genetic elements

Not Essential for survival of Bacteria But makes the
Bacteria Resistant to
antibiotics, and makes them survive
▪Able to produce toxins

Inheritence of characters and variability

 Plasmids
• Plasmids are circular
DNA molecules present
in the cytoplasm of the
Bacteria
• Capable of Autonomous
replication
• Can transfer genes from
one cell to other
• Act as vectors in Genetic
engineering.
• Can also present in
Yeasts

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 Plasmids

• Plasmid seem to be ubiquitous in bacteria, May

encode genetic information for properties
1 Resistance to Antibiotics
2 Bacteriocins production
3 Enterotoxin production
4 Enhanced pathogencity
5 Reduced Sensitivity to
mutagens
6 Degrade complex organic molecules
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 Plasmids

• Can be integrated
with Chromosomal
DNA
• Episomes -
Integrated form of
plasmid with DNA

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Genotypic and Phenotypic variation

• Genome – Sum total of Gene that make up the

genetic apparatus of cell established as Genotype.
• Hereditary constitution of cell this
transmitted to its progeny
• Phenotype – is the physical expression in a
environment. Change according to environment.

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 What is Phenotypic expression

• Exhibit – different phenotypes
• Appearance differs in different situations
• E.g -Typhoid bacilli flagellated normally
• But grown in Phenol agar don't grow flagella So
flagella are lost physical variation
• Lactose fermentation in E.coli dependent on
Beta-Galactosidase
When lactose present - test is positive When
lactose is absent - test turns negative

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 Principles of Genotypic
variations
• Mutations: Change in nucleotide sequence of a gene
• In nature mutation are rare events which occurs at random and
arise spontaneously with no regard to environment conditions
• Isolating a mutant cell is very cumbersome process

• Genotypic by transfer of genes

Transformation
Transduction
Conjugation

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DNA (m RNA ) Proteins X

DNA mRNA p Protein Y

Fluctuation test/ Luria–Delbruck experiment

Results tested by the Luria–Delbruck
experiment
▪ If mutations are induced by the media,(phage) roughly the
same number of mutants are expected to appear on each
plate

▪ If mutations arise spontaneously during cell divisions

prior to plating(before phage contact), each plate will have a
highly variable number of mutants



Replica Plating

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Joshua and Esther Lederberg experiment
(Replica plating technique experiment)

▪Mutation occurs independently

▪Isolation of pure colony of phage resistant bacteria or streptomycin

resistant bacteria is possible without exposure to phage or streptomycin

▪Its now established that bacteria have a hereditory system like higher
organisms

• Bacteria Multiply by asexual binary fission

• Altered Nucleotide sequence in expresses new or
altered characteristics
• Selective value to the organism
• Evolutionary value
• Acquires Antibiotic resistance grows in body
without inhibition
• Become a prominent organism
• Phenotypic variation occurs when genes
changes in response to the environment but
reversible

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Bacterial recombination processes

– 1. Conjugation: direct transfer of DNA from one

bacterial cell to another

–2. Transduction: use of a Bacteriophages (bacterial virus)

to transfer DNA between cells

–3. Transformation: naked DNA is taken up from the

environment by bacterial cells

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 Gene Transfer Processes for

Bacteria and Their Viruses
1. Conjugation
2. Transformation
3. Transduction

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Demonstration of
transformation

Avery, MacLeod, and

McCarty (1944)

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 Transduction

• Generalized involve any segment of DNA

• Restricted when specific Bacteriophages

traduces only a particular genetic trait.
• Transduction effects Plasmids ,and Episomes
• Plasmid transfer induces Penicillin resistance in
Staphylococcus
• Helps Genetic mapping, also in eukaryotic cell
• Helps Genetic Engineering

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 Transduction

• Transduction is
defined as transfer of
portion of DNA from
one bacteria to
another by
Bacteriophages

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 DNA transfer through
Bacteriophages

• When the Phage particle

infects another bacteria
DNA transfer is effected
and the recipient cell
acquires new characters
coded by donor DNA

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 Bacteriophages
• Are viruses that
parasitize bacteria and
consists of Nucleic
acid core and a protein
coat
• A phage particle may
have at its core
besides its own
nucleic acid and a
segment of the Host
DNA

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 Transduction Types

• Two types of Transduction

• 1 Lytic and 2 Lysogenic
• 1 Virulent or Lytic cycle after large number of
progeny are built up inside the host bacterium
ruptures and phages are released

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 Lysogenicity creates new

characters
• Eg - Lysogenic conversion in Diphtheria bacilli
which acquires toxigenicity by lysogenization with
phage beta
• Elimination of phage for toxigenic strain
renders nontoxigenic

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 Gene Transfer Processes for

Bacteria and Their Viruses
1. Conjugation
2. Transformation
3. Transduction

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 Conjugation
Lederberg - Tatum
• A process by which a Donor cell or male cell makes
contact with another cell, the recipient or
Female cell.
• DNA is directly transferable
• Plasmid Carry genetic information necessary for
conjugation to occur.
• Only cell that contain such plasmids can act as donor.
the cell lacking a corresponding plasmid act as
recipient.
• Requires direct contact between donor and recipient

Auxotrophs : microorganism unable to

synthesize all essential nutrients (

Prototrophs: mo synthesize all the

nutrients

 Conjugation
(Lederberg – Tatum)

• A process by which a Donor cell or male cell makes

contact with another cell, the recipient or
Female cell.
• DNA is directly transferable
• Plasmid Carry genetic information necessary for
conjugation to occur.
• Only cell that contain such plasmids can act as donor.
the cell lacking a corresponding plasmid act as
recipient.
• Requires direct contact between donor and recipient

 Conjugation
• The ability to conjugate is conferred by the F plasmid.

• A plasmid is a small circle of DNA that replicates

independently of the chromosome

• Bacterial cells that contain an F plasmid are called

“F+”.

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 Conjugation - Transferring
genes with plasmids

• Plasmids mediating
conjugation carry genes
coding for properties,
of 1-2 microns long
protein appendage
termed Pilus on the
Donor cell

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Mechanism of Transfer I:
Conjugation

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Conjugation

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Simple Conjugation

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Conjugation

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 Pilus helps Conjugation

• Different types of Pilus

are specified by
different types of
plasmids and can help
in aid of plasmid
classification.
• Only one strand of
circular DNA of the
plasmid nicked upon at
a specific site and
passed into a recipient.
• Spread to all other cells.
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 F factor
• Transfer factor that
contains the genetic
information necessary
for synthesis of Sex
Pilus and for self
transfer without any
other identifiable
genetic materials
such as drug
resistance
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F factor helps transformation

• F+ called as Donor bacteria can transform

F - into F+ cell

Can be Episomes able to exist in some cells in the

integrated state in the donor cell chromosome
Can transform chromosomal genes to recruitment with
high frequency are known as Hfr cells
Conversion of F+ cells into Hfr state is reversible.
F factor incorporates some chromosomal genes and is
called as F’
Sexduction The process of transfer of host genes
through F’ factor
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 DNA transfer through
Bacteriophages

• When the Phage

particle infects
another bacteria
DNA transfer is
effected and the
recipient cell
acquires new
characters coded
by donor DNA
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Types of DNA transfer through

Bacteriophages

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 Bacterial Conjugation:
High Frequency Transfer (Her) Cells

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Hfr Conjugation

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Sequence of RTF transmission

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Hfr cell conjugating a Normal cell

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 Genetic Mechanisms of Drug
Resistance
• Bacteria acquire drug resistance through several
Mechanisms
• Mutations
• Genetic transfer
Transformation,
Transduction
Conjugation
Several Biochemical Mechanisms
Decreasing permeability of drugs,
Attaining alternative pathways
Produce enzymes and inactivate drugs

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 TRANSFORMATION

Definition:
▪Transformation is the genetic alteration of a cell resulting from
the uptake and expression of foreign genetic material (DNA). i.e.
the act of putting foreign DNA into a bacterial cell
Occurs in nature, but rarely

Competence
• The ability of some bacteria to take up naked DNA from their
environment.
• It is genetically programmed. Generally, more than a dozen genes
are involved, encoding both regulatory and structural components

• The general steps that occur in natural transformation differ in

Gram-negative and positive bacteria.

 What is Transformation

• Transformation is defined as transfer of Genetic

information through the activity of DNA
• Griffith experiment
Mice injected with Live non capsulated ( R )
Pneumococci
with heat killed capsulated (S) Pneumococci
Lead to death of Mice with isolation of Live
capsulated Pneumococci
It means that some factor from Dead pneumococci
transferred to live non pathogenic Pneumococci

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Griffith Phenomenon

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Steps involved
There are two types of transformation:
1.Natural transformation
2.Artificial transformation
Natural Transformation
• In this case DNA take-up occurs without outside help
• DNA from the environment without requiring special treatment

• About 40 species have been found to be

naturally competent or transformable.
• Examples: Bacillus subtilis, Streptococcus
pneumoniae, Haemophilus influenzae, Neisseria
gonorrhoeae, Helicobacter pylori, Acinetobacter
baylyi, and some species of marine
cyanobacteria.

Following proteins are involved in this process

•ComEA: binds directly extracellular double-stranded DNA

▪The comF genes encode proteins that translocate the DNA into the
cell
▪ComFA provides the energy for translocation of DNA through the
membrane

▪The nucA gene product makes double-strand breaks in extracellular

DNA

▪Other examples are single-stranded-DNA binding protein (SSB)

▪RecA functions in the recombination of transforming DNA with

chromosome DNA.

Natural transformation of Gram negative bacteria

Natural transformation of Gram positive bacteria
 Artificially induced competence

• Bacteria can be sometimes be made competent by

chemical treatments or DNA can be forced into bacteria by a strong
electric field in a process called electroporation

1. Chemical Treatment (with calcium ions)

• Chemically induced transformation is usually inefficient, and only a
small percentage of the cells are ever transformed

• The cells must be plated under conditions, selective for the

transformed cells
• Therefore, the DNA used for the transformation should contain a
selectable gene such as encoding resistance
to an antibiotic.

2. Electroporation

• The bacteria are mixed with DNA and briefly exposed to a strong
electric field.
• The bacteria first be washed extensively in buffer with very low
ionic strength such as distilled water

• The brief electric field across the cellular membranes might create
artificial pore of H2O lined by phospholipid head groups. DNA can
pass through these temporary hydrophilic pores.
• Electroporation requires specialized equipment

Transformation procedure

Plasmid
CaCl2
E. coli cold! E. coli + pAmp

42oC

Recover
E. coli
at 37oC
LB + Amp

Incubate at
37oC overnight
LB + Amp

Applications of Transformation
Bacterial transformation is used:
•To make multiple copies of DNA, called DNA cloning

•To make large amounts of specific human proteins, for

example, human insulin, which can be used to treat people
with Type I diabetes

•To genetically modify a bacterium or other cell.