BACTERIAL GENETICS

Dr. Asiimwe and Dr. Sekyanzi

 Lecture Aim
• To introduce students to the basics of DNA,
RNA, and Genetic Transfer in Bacteria
 DNA
Polymer: Polynucleotide known as a nucleic acid (DNA)
Monomer: Nucleotides
Nucleotide: Pentose sugar, nitrogen base and phosphate
group
 Glycosidic Bond: Between pentose C1 and N-base.

 Phosphodiester bond: Between pentose C5 and phosphate.

 Nucleoside: pentose + nitrogen base only.

Primary structure
 Secondary Structure
• Complimentary Base pairing
• Hydrogen bonds
• Antiparallel strands
• Alpha Helix
Properties of DNA
Polymer
dsDNA (double helix)
ssDNA (hair pins and
stem loops)
Negatively Charged
Circular
Asymmetric and
antiparallel
 RNA
Polymer: Ribonucleic acid
Precursors: Ribonucleoside
triphosphates

Template: DNA

Types of RNA
mRNA, tRNA, rRNA,
tmRNA, sRNA, crRNA
 RNA Structure
Primary: Linear single strand
Secondary: folded (stem loops, helices and
hair pins)
Tertiary structures: requires metal ions
Central Dogma
Genetic Transfer & Recombination in
Bacteria
 Genetic Transfer
• Definition; mechanism by which DNA is transferred from a
donor to a recipient.
• Once donor DNA is inside the recipient, crossing over occurs.
– The result is a recombinant cell that has a genome different
from either the donor or the recipient.
Gene transfer mechanisms in bacteria
– Vertical transfer: parent to offspring, cell division; obeys
Dogma laws
– Horizontal gene transfer: the gist of bacterial genetics
• In bacteria genetic transfer can happen three ways:
– Transformation
– Transduction
– Conjugation
• Remember that a recombination event must occur after transfer in order that the change in the
genome be heritable
 Transformation
• Genetic recombination in which a DNA fragment from a
dead, degraded bacterium enters a competent recipient
bacterium and it is exchanged for a piece of the recipient's
DNA.
• Involves 4 steps;

1. A donor bacterium dies and is degraded 2. A fragment of DNA from the dead donor
bacterium binds to DNA binding proteins on the
cell wall of a competent, living recipient
bacterium

3. The Rec A protein promotes genetic

exchange between a fragment of the
donor's DNA and the recipient's DNA
4. Exchange is complete
 GRIFFITH’S EXPERIMENT
Transformation was first demonstrated by Frederick Griffith in 1928.  Griffith
(knowing nothing about DNA) concluded that some "transforming principle"
was passed from the dead smooth (pathogenic) strain to the live rough (non-
pathogenic) strain.
 Transduction
• Genetic recombination in which a DNA fragment is transferred
from one bacterium to another by a bacteriophage
• Bacteriophage (phage) are obligate intracellular parasites that
multiply inside bacteria by making use of some or all of the host
biosynthetic machinery (i.e., viruses that infect bacteria)

Structure of T4 bacteriophage
 Bacteriophage structure
• Most bacteriophage have tails

• The size of the tail varies.

• It is a tube through which the nucleic acid is injected as a result of

attachment of the bacteriophage to the host bacterium
• In the more complex phages the tail is surrounded by a contractile sheath for
injection of the nucleic acids
• Many bacteriophages have a base plate and tail fibers

• Some have icosahedral capsids

• M13 has a helical capsid

 Transduction
There are two types of transduction:
a. generalized transduction:
– A DNA fragment is transferred from one bacterium to another
by a lytic bacteriophage that is now carrying donor bacterial
DNA due to an error in maturation during the lytic life cycle.
– random host-cell DNA is carried by the defective (tranducing)
phage
b. specialized transduction:
-A DNA fragment is transferred from one bacterium to another
by a temperate bacteriophage that is now carrying donor
bacterial DNA due to an error in spontaneous induction during
the lysogenic life cycle
-
-Some bacteria, when infected by a specialized transducing
phage, become pathogenic, since the genes for virulence are
carried by the phage.
 Seven steps in Generalised Transduction

1. A lytic bacteriophage 2. The bacteriophage genome enters the

adsorbs to a susceptible bacterium. The genome directs the bacterium's
bacterium. metabolic machinery to manufacture
bacteriophage components and enzymes

3. Occasionally, a bacteriophage head

or capsid assembles around a fragment 4. The bacteriophage are released.
of donor bacterium's nucleoid or
around a plasmid instead of a phage
genome by mistake.
Seven steps in Generalised Transduction

5. The bacteriophage carrying the donor

bacterium's DNA adsorbs to a recipient bacterium

6. The bacteriophage inserts the donor

bacterium's DNA it is carrying into the recipient
bacterium .

7. The donor bacterium's DNA is exchanged for some

of the recipient's DNA.
Six steps in Specialised Transduction
1. A temperate
bacteriophage adsorbs to a
susceptible bacterium and
injects its genome .
4. As the bacteriophage
replicates, the segment of
bacterial DNA replicates as
part of the phage's genome.
Every phage now carries that
segment of bacterial DNA.
2. The bacteriophage
inserts its genome into the
bacterium's nucleoid to
become a prophage.
5. The bacteriophage
adsorbs to a recipient
bacterium and injects its
genome.
3. Occasionally during spontaneous
induction, a small piece of the
donor bacterium's DNA is picked up
as part of the phage's genome in
place of some of the phage DNA
which remains in the bacterium's
nucleoid.
6. The bacteriophage genome
carrying the donor bacterial
DNA inserts into the recipient
bacterium's nucleoid.
 Bacterial Conjugation

Bacterial Conjugation is genetic recombination in which

there is a transfer of DNA from a living donor bacterium to a
recipient bacterium. Often involves a sex pilus.

• The 3 conjugative processes

a. F+ conjugation
b. Hfr conjugation
c. Resistance plasmid conjugation
 F+ Conjugation Process

• Genetic recombination in which there is a transfer of an

F+ plasmid (coding only for a sex pilus) but not chromosomal
DNA from a male donor bacterium to a female recipient
bacterium.

• Involves a sex (conjugation) pilus. Other plasmids present

in the cytoplasm of the bacterium, such as those coding for
antibiotic resistance, may also be transferred during this
process.
 The 4 stepped F+ Conjugation
1. The F+ male has an F+ plasmid coding
for a sex pilus and can serve as a genetic
donor
3. The sex pilus retracts and a bridge is
created between the two bacteria. One
strand of the F+ plasmid enters the
recipient bacterium
2. The sex pilus adheres to an F- female
(recipient). One strand of the F+ plasmid
breaks
4. Both bacteria make a complementary
strand of the F+ plasmid and both are
now F+ males capable of producing a sex
pilus. There was no transfer of donor
chromosomal DNA although other
plasmids the donor bacterium carries
may also be transferred during F+
conjugation.
 Hfr Conjugation
• The "Hfr" or"high frequency recombination"state describes
the situation where fragments of chromosomal DNA from a
male donor bacterium are transferred to a female recipient
bacterium following insertion of an F+ plasmid into the
nucleoid of the donor bacterium. Involves a sex (conjugation)
pilus.

• Both plasmid genes and chromosomal genes are transferred

 5-step Hfr Conjugation

1. An F+ plasmid inserts into the donor 2. The sex pilus adheres to an F- female
bacterium's nucleoid to form an Hfr male. (recipient). One donor DNA strand breaks
in the middle of the inserted F+ plasmid.

3. The sex pilus retracts and a bridge forms between

the two bacteria. One donor DNA strand begins to
enter the recipient bacterium. The two cells break
apart easily so the only a portion of the donor's DNA
strand is usually transferred to the recipient
bacterium.
5-step Hfr Conjugation
4. The donor bacterium makes a
complementary copy of the remaining DNA
strand and remains an Hfr male. The
recipient bacterium makes a
complementary strand of the transferred
donor DNA.

5. The donor DNA fragment undergoes genetic

exchange with the recipient bacterium's DNA.
Since there was transfer of some donor
chromosomal DNA but usually not a complete
F+ plasmid, the recipient bacterium usually
remains F-
 Resistant Plasmid Conjugation
Genetic recombination in which there is a transfer of an R
plasmid (a plasmid coding for multiple antibiotic resistance and
often a sex pilus) from a male donor bacterium to a female
recipient bacterium. Involves a sex (conjugation) pilus

1. The bacterium with an R-plasmid is multiple

antibiotic resistant and can produce a sex pilus
(serve as a genetic donor).

2. The sex pilus adheres to an F- female

(recipient). One strand of the R-plasmid breaks.
Resistant Plasmid Conjugation

3. The sex pilus retracts and a bridge is created

between the two bacteria. One strand of the R-
plasmid enters the recipient bacterium.

4. Both bacteria make a complementary strand

of the R-plasmid and both are now multiple
antibiotic resistant and capable of producing a
sex pilus.
 Significance

• Increase in drug resistance when one bacterial cell acquires

resistance, and the resistance genes are transferred to other
species.
 BACTERIAL TRANSPOSONS

1. “Transposable elements or Mobile DNA ;are pieces of DNA

that can move from one location on the chromosome to
another, from plasmid to chromosome or vice versa or
from one plasmid to another.
a. Sometimes a copy is made and the copy moves
b. Insertion requires target DNA sequences

2. These mobile segments of DNA are sometimes called

"jumping genes"
 BACTERIAL TRANSPOSONS cont’

In the process, they may

• Cause mutations
• Increase (or decrease) the amount of DNA in the
genome.
• Promote genome rearrangements.
• Regulate gene expression.
• Induce chromosome breakage and rearrangement.
 Types of transposons

There are two distinct types of transposons:

a. DNA transposons
Transposons consisting of only DNA that moves directly from
place to place

b. Retrotransposons
First transcribe the DNA into RNA and the use Reverse
transcriptase to make a DNA copy of the RNA to insert in a
new location
 Transposable Genetic Elements

• Definition: Segments of DNA that are able to move from one

location to another
• Properties
– “Random” movement
– Not capable of self replication
– Transposition mediated by site-specific recombination
• Transposase
– Transposition may be accompanied by duplication
 Types of Transposable Genetic Elements

• Insertion sequences (IS)

– Definition: Elements that carry no other genes
except those involved in transposition
– Nomenclature - IS
– Structure ABCDEFG Transposase GFEDCBA
– Importance
• Mutation
•Plasmid insertion
 Transposons (Tn)

a. Definition: Elements that carry other genes except

those involved in transposition
b. Nomenclature - Tn
c. Structure

IS Resistance Gene(s) IS

IS Resistance Gene(s) IS

d. Importance
• Antibiotic resistance
 Insertion sequences
1. Insertion sequences – IS1 and IS186, present in the 50-kb
segment of the E. coli DNA, are examples of DNA
transposons. Single E. coli genome may contain 20 of them.

2. Most of the sequence is taken by one or two genes for

transposase enzyme that catalyses transposition.

3. IS elements transpose either replicatively or

conservatively.
 Bacterial IS element

a. Central region encodes for one or two enzymes required for transposition. It is
flanked by inverted repeats of characteristic sequence.

b. The 5’ and 3’ short direct repeats are generated from the target-site DNA during
the insertion of mobile element.

c. The length of these repeats is constant for a given IS element, but their sequence
depends upon the site of insertion and is not characteristic for the IS element.

d. Arrows indicate orientation.

 Mechanism of transposition
Two distinct mechanisms of transposition:
a. Replicative transposition – direct interaction between the
donor Transposon and the target site, resulting in copying of
the donor element
b. Conservative transposition – involving excision of the
element and reintegration at a new site.
1. Replicative transposition 2.Non-replicative (conservative)
transposition
Copy of transposon sequence
- Cannot copy transposons sequence
- Transposition by cut and paste
Transposase enzyme cut target DNA model

- Cut transposons sequence from

Transposition donor molecule attach to target
Duplication of target sequence site Ex. IS10, Tn10
 Diseases
Transposons are mutagens. They can damage the genome of their host
cell in different ways:

a. A transposon or a retroposon that inserts itself into a functional gene

will most likely disable that gene.

b. After a transposon leaves a gene, the resulting gap will probably not be
repaired correctly.

c. Multiple copies of the same sequence, such as Alu sequences can hinder
precise chromosomal pairing during mitosis and meiosis, resulting in
unequal crossovers, one of the main reasons for chromosome
duplication.
d. Diseases caused by transposons include
-hemophilia A and B
-severe combined immunodeficiency
-Porphyria
-Cancer
-Duchenne muscular dystrophy
 Applications

• Researchers use transposons as a means of

mutagenesis.

• To identifying the mutant allele.

• To study the chemical mutagenesis methods.

• To study gene expression.

 Recombinant DNA technology
• Recombinant DNA is any DNA molecule
composed of sequences derived from different
sources.

• Recombinant DNA is also sometimes referred to

as "chimera."

• The most common recombinant process involves

combining the DNA of two  different organisms. 
 Recombinant DNA technology
• The procedure involves extracting and cutting up
DNA from a donor genome into fragments
containing from one to several genes and allow
these fragments to insert themselves individually
into opened-up small autonomously replicating
DNA molecules such as bacterial plasmids,
phages
• These small molecules act as carriers,
or vectors, for the DNA fragments.
The vector molecules with their inserts are
called recombinant DNA 
 Recombinant DNA technology
Vector DNA fragment
↓
Recombinant DNA
↓
Replication of recombinant DNA within host cells
↓
Isolation, sequencing, and manipulation
of purified DNA fragment
Restriction Enzymes and DNA Ligases
During DNA cloning discrete, small regions of an
organism’s DNA that constitute specific genes are required.

In addition, only relatively small DNA molecules can be

cloned in any of the available vectors.

For these reasons, the very long DNA molecules that

compose an organism’s genome must be cleaved into
fragments that can be inserted into the vector DNA.

Two types of enzymes—Restriction enzymes and DNA

ligases.
 Cutting DNA Molecules into Small
Fragments: Restriction
Enzymes used are endonucleases produced by
bacteria that typically recognize specific 4- to 8-
bp sequences, called restriction sites, and then
cleave both DNA strands at this site.

Restriction sites commonly are short

palindromic sequences; that is, the restriction-
site sequence is the same on each DNA strand
when read in the 5 → 3 direction.
 Cutting DNA Molecules into Small
Fragments
Many restriction enzymes make staggered cuts in the two
DNA strands at their recognition site, generating fragments
that have a single-stranded “tail” at both ends .

The tails on the fragments generated at a given restriction site

are complementary to those on all other fragments generated
by the same restriction enzyme.

“sticky ends,” can transiently base-pair with those on other

DNA fragments generated with the same restriction enzyme.

A few restriction enzymes, such as AluI and SmaI, cleave both

DNA strands at the same point within the restriction
Cutting DNA Molecules into Small
Fragments
A few restriction enzymes, such as AluI and SmaI, cleave both
DNA strands at the same point within the restriction
site, generating fragments with “blunt” ends in which all the
nucleotides at the fragment ends are base-paired to
nucleotides in the complementary strand.

Many other restriction enzymes also produce

fragments with sticky ends.

A given restriction enzyme will cut the DNA from a particular

source into a reproducible set of fragments called restriction
fragments.
Inserting DNA Fragments into Vector
DNA
DNA Fragments with either sticky ends or blunt ends
can be inserted into vector DNA with the aid of DNA
ligases.

DNA ligase catalyzes the end-to-end joining (ligation)

of short fragments of DNA, called Okazaki fragments.
Transformation
 Phage Introduction

Phage introduction is the process of transfection, which

is equivalent to transformation, except a phage is used
instead of bacteria.

In vitro packagings of a vector is used. This uses lambda

or MI3 phages to produce phage plaques which contain
recombinants. 

The recombinants that are created can be identified by

differences in the recombinants and non-recombinants
using various selection methods. 
 Phage Introduction
Bacteriophage lambda, which infects E. coli, has been
widely used as a cloning vector.

Although lambda DNA circularizes for replication and

insertion into the E. coli chromosome, the DNA inside
the phage particle is linear
Only DNA molecules of between 37 and 52 kb can be
stably packaged into the head of the lambda particle.

Small fragments of extra DNA may be inserted into the

lambda genome without preventing packaging.
 Phage Introduction
The middle region (~15 kb) of the lambda genome
is non-essential and may be replaced with
approximately 23 kb of foreign DNA.

In vitro packaging is done where a mixture of

lambda proteins is mixed with the recombinant
lambda DNA in vitro to form phage particles.

E.coli is infected with phage particles and it is

cloned.