Bacterial genetics

Mutation and Recombination

Kennedy Kassaza
Lecturer –Dept. of Microbiology
Mbarara University of Science and Technology
 Bacterial Genetics

• Genetics is the study of genes including the structure of genetic materials, what
information is stored in the genes, how the genes are expressed and how the
genetic information is transferred
- Introns - non coding sequences on a gene.
- Exons - coding sequences on a gene translated into gene products

- Bacterial genetics is used as a model to understand DNA replication, genetic

characters, their changes & transfer to next generations
Nucleic Acids
• DNA (Deoxy ribonucleic acid) : stores information for protein synthesis.
• RNA (ribonucleic acid) : transcription & translation of information for protein
synthesis.
Central Dogma : DNA – RNA - Protein.
 Structure Of DNA

• Proposed by Watson & Crick.

• Double helix model.
• Composed of 2 chains of
polypeptides, each chain has a
backbone of deoxyribose sugar
and phosphate residues
arranged alternately.
• 4 nitrogenous bases: Adenine
(A) Purine Guanine (G)
Thymine(T) Pyrimidine Cytosine
(C)
Structure Of RNA

• Structurally similar to DNA, except for 2 major differences:

- ribose sugar uracil in place of thymine.
3 types of RNA
- m RNA (messenger RNA)
- t RNA ( transfer RNA )
- r RNA ( ribosomal RNA )
Genetic Information

• In Bacteria Chromosome Carries

properties like virulence,
pathogenicity & resistance

• Plasmids are Extrachromosomal

genetic material in the
cytoplasm

• Bacteriophage: Replicate
independently
PLASMIDS

• Circular DNA molecules

• Important vectors in genetic engineering
• EPISOME Plasmid DNA integrated with chromosomal DNA.
Types of plasmids
- R plasmid (drug resistance): RTF* + r determinant
- F plasmid (maleness )

* Resistance Transfer Factor

Genotypic & Phenotypic
Variations
• Genotype – genetic constitution of a cell that is transmitted to its
progeny
• Phenotype – physical expression of the genotype in a given
environment
Variations
1. Phenotypic variations – influenced by the environment temporary &
not heritable
2. Genotypic variations – Not influenced by the environment Stable &
heritable
Mechanisms Of Genetic
Variations
• Mutation
• Transfer or exchange of genetic material. Occurs in the following
ways:
1. Transformation
2. Transduction
3. Conjugation
4. Lysogenic conversion
5. Transposition
Mutation….
• Random, undirected heritable variation Caused by a change in the
nucleotide base sequence of the DNA
• Types of mutation:
1. Point mutation
2. Frame shift mutation
3. Lethal mutation
4. Suppressor mutation Mutagens - Agents which can induce mutation
e.g. UV rays,
5 bromouracil, alkylating agents, etc.
1. Point Mutation

• Cause
- due to addition, deletion or substitution of one or more bases.
Types
- Transition : a purine base is replaced by a purine base or a pyrimidine
base is replaced by another pyrimidine base. Most common type.
- Transversion : substitution of a purine base by a pyrimidine base & vice
versa
 Results of point mutation
• A sense mutation
A single substitution
mutation which results
in a new codon still
coding for the same
amino acid

• A mis-sense mutation
A single substitution
mutation which results
in one wrong codon
and, therefore, one
wrong amino acid.
 A non-sense mutation
• A single substitution
mutation which
results in
transcription of a
stop or nonsense
codon resulting in
the termination of
polypeptide chain
2. Frame Shift Mutation

• Cause - Deletion or
insertion of a base -
changes all of the
codons downstream
from the change
3. Lethal Mutation

• Mutation which resulting involve vital functions in the death of the

organism
– nonviable mutation. A conditional lethal mutant may be able to live under
certain conditions
– permissive conditions. Commonest type of conditional mutant is the
temperature sensitive (t s) mutant which is able to live at the permissive
temperature of 35 °C but not at the restrictive temp (39 °C).
 Suppressor Mutation
• Reversal of a mutant phenotype by
another mutation at a position on
the DNA, distinct from that of the
original mutation.

Lederberg & Tatum (1946)

Experiment demonstrating
recombination in E. coli.
Recombination of 2 complimentary
auxotrophs gives rise to a strain that
can synthesize all nutrients
Bernard Davis
experiment
demonstrated that
physical contact is
for bacterial
recombination. .
1. Conjugation
• First described by Lederburg & Tatum in 1946 in a strain of E.coli
called K12.
• A donor or male bacterium passes DNA directly to a recipient or
female bacterium by a conjugation tube (sex pili). The female
bacterium attains donor status & in turn can conjugate with other
female cells.
• Maleness is determined by the presence of a plasmid which codes for
sex pili.
• The plasmid is called the sex factor or fertility factor (F factor)
• R (resistance) factor can also be transferred by conjugation
Conjugation-transfer of the sex factor F:
1. William Hayes (1953) demonstrated that genetic exchange in E. coli
occurs only in one direction.
2. Genetic transfer is mediated by sex factor F.
3. Donor is F+ and recipient is F-.
4. F is a self-replicating, circular DNA plasmid (1/40 the size of the main
chromosome).
5. F plasmid contains an origin sequence (O), which initiates DNA
transfer. It also contains genes for hair-like cell surface (F-pili or sex-pili),
which aid in contact between cells..
 F factor and Conjugation

• F (fertility) factor is a
conjugative plasmid
transferred from cell to cell
by conjugation
• F factor is an episome =
genetic element that can
insert into chromosome or
replicate as circular
plasmid
• The F plasmid is a low-
copy-number plasmid ~100
kb in length, and is present
in 1–2 copies per cell
• It replicates once per cell
cycle and segregates to
both daughter cells in cell
division
 Transfer pf
F-factor

• 1. No conjugation can
occur between cells of
the same mating type.
• 2. Conjugation begins
when the F plasmid is
nicked at the origin, and
a single strand is
transferred using the
rolling circle
mechanism.
• 3. When transfer is
complete, both cells are
F + doublestranded
 Conjugation of high-
frequency Transfer of F- factor
recombinant
strains:
1. No chromosomal DNA is transferred by
standard sex factor F.
2. Transfer of chromosome DNA is facilitated
by special strains of F + integrated into the
bacteria chromosome by crossing over.
3. Hfr strains = high frequency recombination
strains.
4. Discovered by William Hayes and Luca
Cavalli-Sforza. 5. Hfr strains replicate F
factor as part of their main chromosome.
 Transfer of Hfr F+ factor

1. Conjugation in Hfr strains begins

when F+ is nicked at the origin, and
F+ and bacteria chromosomal DNA
are transferred using the rolling
circle mechanism.
2. Complete F+ sequence (or
complete chromosomal DNA) is
rarely transferred (1/10,000)
because bacteria separate
randomly before DNA synthesis
completes.
3. Recombinants are produced by
crossover of the recipient
chromosome and donor DNA
containing F+.
 Excision of the F+ factor also occurs
spontaneously at low frequency.

1. Begin with Hfr cell

containing F+.
2. Small section of host
chromosome also may
be excised, creating an
F’ plasmid.
3. F’ plasmid is named for
the gene it carries, e.g.,
F’ (lac)
 2. Transformation (Griffith, 1928)
• Transfer of genetic
information by free DNA.
i.e. by direct uptake of
donor DNA by the recipient
DNA.
• Live non capsulated (R)
pneumococci + heat killed
capsulated (S) pneumococci
- Injected into mice
- Death of mice
• Live capsulated
pneumococcus isolated
from the blood of mice
Bacterial
Transformation
is the process of DNA
uptake by the
bacteria from the
surrounding
environment.
The cells that have
the ability to uptake
DNA are known as
competent cells.
This process was first
reported
in Streptococcus
pneumonia by Griffith.
Transformation

1. Unidirectional transfer of
extracellular DNA into cells,
resulting in a phenotypic change
in the recipient.
2. First discovered by Frederick
Griffith (1928).
3. DNA from a donor bacteria is
extracted and purified, broken
into fragments, and added to a
recipient strain.
4. Donor and recipient have
different phenotypes and
genotypes.
5. If recombination occurs, new Transformation:
recombinant phenotypes appear. 1. Bacteria vary in their ability to take up DNA.
2. Bacteria such as Bacillus subtilis take up DNA naturally.
3. Other strains are engineered (i.e., competent cells).
4. Competent cells are electrophoresed or treated chemically to induce E. coli to take
up extracellular DNA.
3. Transduction

1. Bacteriophages (bacterial viruses) transfer genes to bacteria (e.g., T2,

T4, T5, T6, T7, and λ).
- Generalized transduction transfers any gene.
- Specialized transduction transfers specific genes.
2. Phages typically carry small amounts of DNA, ~1% of the host
chromosome.
3. Viral DNA undergoes recombination with homologous host
chromosome DNA.
4. Most widely used mechanism of gene transfer among prokaryotes
 Transduction

• Transfer of a portion of the

DNA from one bacterium to
another by a bacteriophage.
- Packaging error within the
infected bacteria during the
assembly of progeny phages –
presence of a segment of host
DNA along with the phage
nucleic acid in the core of
phage Infection of another
bacterium
- Transfer of host bacterial
DNA to the new bacterium
Acquisition of new
characteristics coded by the
donor DNA.
 Life cycle of phage λ Fig. Generalized transduction of E. coli by phage P1
Fig. Life cycle of phage λ
 Lysogenic Conversion

• Phage DNA itself is the

new genetic element.
Bacteriophages – 2
Types of life cycle
• Lytic or virulent cycle –
progeny viruses build
up inside host
bacterium, which
rupture to release
them.
• Temperate or non-lytic
or lysogenic cycle – host
bacterium is unharmed.
 Lysogeny
Process of Conjugation
• Lysogenic bacteria
• Prophage behaves as
an additional segment
of bacterial
chromosome, coding
for new
characteristics. This
process by which
prophage confers
genetic information to
a bacterium is called
Lysogenic conversion .
4. Transposon (Jumping Genes,
Barbara McClintock)
• DNA segment that can
move between
chromosome & plasmids
• Insertion of transposon into
a functional gene would
destroy the function of the
gene (internal mutagenic
agents)
• Transposons are not self
replicative, they depend on
chromosomal or plasmid
DNA for replication